Alginate oligomers for use in overcoming multidrug resistance in bacteria

ABSTRACT

The invention provides a method of overcoming resistance to at least one antibiotic in a multidrug resistant bacterium, said method comprising contacting said bacterium with an alginate oligomer together with the antibiotic. The multidrug resistant bacterium may be on an animate or inanimate surface and both medical and non-medical uses and methods are provided. In one aspect the invention provides an alginate oligomer for use together with at least one antibiotic in treating a subject infected, suspected to be infected, or at risk of infection, with a multidrug resistant bacterium to overcome resistance to the antibiotic in said multidrug resistant bacterium. In another aspect the method can be used to combat contamination of a site with multidrug resistant bacteria, e.g. for disinfection and cleaning purposes.

The present invention relates to alginate oligomers for use togetherwith (or in combination or conjunction with) an antibiotic to overcome(in the sense of reducing) resistance to the antibiotic in a multidrugresistant (MDR) bacterium. Whilst a principal and important use of thepresent invention is in the treatment or prevention of bacterialinfections with MDR bacteria, namely a medical use, the invention alsoencompasses such use of alginate oligomers in non-medical settings (e.g.in vitro). The invention thus provides alginate oligomers for usetogether with (or in combination or conjunction with) an antibiotic forthe treatment or prevention of an MDR bacterial infection in a subject,or for combating MDR resistant bacteria in vitro (for example incombating the microbial contamination (i.e. colonisation) of an abioticsite with MDR bacteria).

Ever since antibiotics were first used it was appreciated that bacteriacould display intrinsic resistance to these drugs or could developresistance to these drugs. Resistance of a bacterium to an antibioticcan be viewed as a substantially greater tolerance, or reducedsusceptibility, to the antibiotic compared to a sensitive bacterium or atypical or a wild type version of the bacterium. In some cases abacterium can be completely unaffected by exposure to an antibiotic. Inthis instance the bacterium can be considered fully resistant to thatantibiotic.

Multidrug resistance (MDR) in bacteria describes the situation where abacterium is resistant to at least three classes of drugs, specificallyin the context of bacteria, at least three classes of anti-microbial (ormore specifically anti-bacterial) agents, and particularly in thecontext of the present invention, at least three classes of antibiotics.Antibiotics in one class are functionally unrelated, structurallyunrelated, or both, to antibiotics in a different class. MDR in bacteriais thus often termed multiple anti-bacterial drug resistance or multipleantibiotic resistance. The terms are used interchangeably in the art andherein. Bacteria displaying multidrug resistance phenotypes (or multipleantibacterial/antibiotic drug resistance phenotypes) are referred to asMDR bacteria (or sometimes MAR bacteria). Again, these terms are usedinterchangeably in the art and herein.

Antibiotic resistance mechanisms are numerous. For instance, resistancemay arise from impermeability mechanisms which physically prevent theantibiotic reaching its site of action in or on the bacterium; effluxmechanisms which prevent effective amounts of the antibiotic reachingits site of action in or on the bacterium by rapidly removing theantibiotic from the bacterium; metabolic mechanisms which breakdown theantibiotic or convert the antibiotic into a harmless (or less harmful)compound, or a compound more easily excreted; bypass mechanisms in whichthe bacterium uses alternative pathways to those inhibited by theantibiotic; or through the bacterium having a form of the antibiotictarget (e.g. enzyme) that is less sensitive to the antibiotic or nothaving the target at all.

Resistance to a particular antibiotic or class of antibiotic may beintrinsic to the bacterium, but it can also be developed or acquired.Generally intrinsic resistance may be seen to a particular type or classof antibiotic, but the number of different antibiotic classes to whichresistance is seen is usually restricted. Resistance to numerous classesof antibiotics (including to multiple classes of antibiotics, which isdefined herein as at least three classes of antibiotics) may be anacquired (or developed) phenomenon, but this is not exclusively thecase. Thus, in the case of MDR bacteria, the bacteria may acquire ordevelop resistance to particular antibiotic classes (e.g., to one ormore or two or more classes, for example additional classes, or to 3 ormore classes), or in certain cases the bacteria may be intrinsicallyresistant to multiple classes. The resistant phenotype of MDR bacteriacan differ from typical or wild type bacteria, but certain bacteria canbe considered MDR on account of their intrinsic resistance profile, e.g.Burkholderia species including Burkholderia cepacia, Burkholderiamallei, and Burkholderia pseudomallei.

Development (or acquisition) of resistance can be through mutation. Forinstance, this may involve changes in the structure of the target of theantibiotic that reduces the sensitivity of the target to the antibiotic.It can also be a mutation in a pathway involved in the regulation of thecellular machinery involved in the metabolism or efflux of theantibiotic. It can also be a mutation in the constituents of the outerlayers (e.g. the membranes/walls) of the bacterium that effects thepermeability of the antibiotic into the bacterium. In some instancesmultiple mutations must accumulate in order for a bacterium to becomeresistant to a particular antibiotic or class thereof.

Development of resistance can also be through the transfer of aresistance mechanism from another organism, e.g. another bacterium (thisis sometimes referred to as acquired resistance, but as used herein theterm “acquired resistance” includes any means or mechanism by which theresistance arises, including by transfer or by mutation). This isusually, although not exclusively, though the transfer from organism toorganism of mobile nucleic acids encoding the resistance mechanism (e.g.β-lactamase).

As a consequence of the inherent selective pressure antibiotics exert ona bacterial population, the use of antibiotics selects for resistantmembers of that population. The sequential use of different antibioticsin a treatment regime can therefore give rise to MDR bacteria.

Many MDR species and strains of bacteria exist today. Bacterial generafrom which MDR species and strains pose significant problems for humanand animal health include, but are not limited to Pseudomonas,Acinetobacter, Burkholderia, Klebsiella, Providencia, and Staphylococcus

Pseudomonas is a genus of strictly aerobic, gram-negative bacteria ofrelatively low virulence. Nevertheless, Pseudomonas species can act asopportunistic pathogens and infections have been reported withPseudomonas aeruginosa, Pseudomonas oryzihabitans, Pseudomonas luteola,Pseudomonas anguilliseptica and Pseudomonas plecoglossicida.

P. plecoglossicida and P. anguilliseptica are fish pathogens. P.oryzihabitans can be a human pathogen causing peritonitis,endophthalmitis, septicemia and bacteriaemia. Similar infections can becaused by P. luteola. The majority of Pseudomonas infections in humansare, however, caused by P. aeruginosa.

P. aeruginosa is a widespread and extremely versatile bacteria that canbe considered a part of the natural flora of a healthy subject and iscapable of colonising most man-made environments. This ubiquity andversatility has seen colonisation of healthcare environments by P.aeruginosa. Problematically, the same versatility enables P. aeruginosato act as an opportunistic human pathogen in impaired subjects, mostcommonly immunocompromised patients (e.g. those with, cystic fibrosis orAIDS) and patients with a compromised barrier to infections (e.g. thosewith chronic wounds and burns and those with in-dwelling medical devicessuch as intravenous lines, urinary catheters, dialysis catheters,endotracheal tubes).

P. aeruginosa infection can affect many different parts of the body, butinfections typically target the respiratory tract, the GI tract, theurinary tract and surface wounds and burns and in-dwelling medicaldevices. This problem is compounded by the presence of intrinsicresistance to many of the β lactam antibiotics. Acquired resistance ofcertain strains to further antibiotics is also being reported. Theability of certain strains of P. aeruginosa to form biofilms addsfurther to these problems because biofilm-dwelling bacteria are oftenmore resistant to anti-microbials than their non-biofilm counterparts.As such, there is an urgent need for safe and effective treatments forPseudomonas infections and contamination and, in particular, treatmentsthat overcome antibiotic resistance, particularly β-lactam resistance,in Pseudomonas species.

Burkholderia is a genus of gram-negative, motile, obligate aerobic,non-fermenting rod-shaped bacteria. Burkholderia species are widelydistributed in nature and include animal and plant pathogens.Burkholderia cepacia is emerging as a human pathogen of note. B. cepaciahas been reported to have caused necrotizing pneumonia,ventilator-associated pneumonia, bacteraemia, and infections of theskin, soft tissue, bloodstream, respiratory tract, and urinary tract incystic fibrosis patients and hospitalised patients. Burkholderia cepaciais a part of a group of at least nine different species forming theBurkholderia cepacia complex (BCC), including B. multivorans, B.cenocepacia, B. vietnamiensis, B. stabilis, B. ambifaria, B. dolosa, B.anthina, and B. pyrrocinia

Burkholderia pseudomallei, is the causative agent of melioidosis, apotentially fatal community-acquired infectious disease endemic tosoutheast Asia, Taiwan and northern Australia. Cases have also beendescribed in China, India, Central and South America, the Middle East,and several African countries. Incidences of the disease amongstservicemen engaged in conflicts in these areas have been reported andspread of the diseases back to the country of origin of these servicemenhas been noted and is a consequence of the fact that relapses are commonand the disease can remain latent for long periods before clinicalmanifestation.

Burkholderia mallei, is the causative agent of glanders, an infectiousdisease that primarily affecting horses, mules and donkeys, but it hasbeen reported in other animals, e.g. dogs, cats and goats, and inparticular, transmission to humans can occur. Transmission from theanimal to human typically occurs by direct contact through skinabrasions, nasal and oral mucosal surfaces, or by inhalation.

Problematically, pathogenic Burkholderia species often display intrinsicresistance to multiple antibiotics and antibiotic classes (e.g. one ofmore of the aminoglycosides, β-lactams and macrolides) and persistencein betadine (a topical antiseptic used commonly in hospitals) has beennoted. Acquired resistance of certain strains to further antibiotics isalso being reported. As such, there is an urgent need for safe andeffective treatments for Burkholderia infections and contamination and,in particular, treatments that overcome antibiotic resistance,particularly, β-lactam and macrolide resistance, in Burkholderiaspecies.

Providencia is a genus of gram-negative bacilli that are responsible fora wide range of human infections. Providencia infections are usuallynosocomial and are found predominantly in the urinary tract, often as aconsequence of catheterisation. Providencia infections are alsoassociated with gastroenteritis and bacteraemia and surface infectionsof chronic wounds and burns. They represent an emerging problem becauseof the increasing prevalence of strains with β-lactam antibioticresistance due to the spread amongst Providencia populations ofextended-spectrum beta-lactamase (ESBL).

Providencia species include Providencia stuartii, Providencia sneebia,Providencia rettgeri, Providencia rustigianii, Providencia heimbachae,Providencia burhodogranariea and Providencia alcalifaciens. Providenciaspecies have been found in soil, water and sewage and in multiple animalreservoirs. Examples of Providencia infections in animals includeneonatal diarrhoea in cattle due to P. stuartii infection and enteritiscaused by P. alcalifaciens infection in dogs. P. rettgeri has beenisolated in crocodiles with meningitis/septicaemia and in chickens withenteritis. P. heimbachae has been isolated in penguin faeces and abortedbovine foetuses.

In humans, Providencia species have been isolated from urine, stool, andblood, as well as from sputum, skin, and wound cultures. P. stuartii isfrequently isolated in patients with indwelling urinary catheters and isknown to persist in the urinary tract after bladder access is attained.P. stuartii can give rise to septicaemia, and commonly this is secondaryto the infection of the urinary tract. P. stuartii has also beenreported as the etiology of infective endocarditis. P. rettgeri has beenreported to be a cause of ocular infections, including keratitis,conjunctivitis, and endophthalmitis. P. alcalifaciens, P. rettgeri, andP. stuartii have also been implicated in gastroenteritis.

Providencia infections with antimicrobial resistance patterns areincreasing and ESBL-positive P. stuartii is an increasing problem inhospitalized patients. As such, there is an urgent need for safe andeffective treatments for Providencia infections and contamination and,in particular, treatments that overcome antibiotic resistance,particularly β-lactam resistance, in Providencia species.

Acinetobacter is a genus of bacteria that are strictly aerobicnon-fermentative gram-negative bacilli. Acinetobacter species are widelydistributed in nature and can survive for long periods of time on wet ordry surfaces. Acinetobacter species are considered to be non-pathogenicto healthy subjects, but it is becoming increasingly apparent thatAcinetobacter species persist in hospital environments for a long periodof time and can be responsible for nosocomial infections in compromisedpatients. Acinetobacter baumannii is a frequent cause of nosocomialpneumonia, especially of late-onset ventilator associated pneumonia andit can cause various other infections including skin and woundinfections, bacteraemia, and meningitis. Acinetobacter lwoffii has alsobeen associated with meningitis. Other species including Acinetobacterhaemolyticus, Acinetobacter johnsonii, Acinetobacter junii,Acinetobacter radioresistens, Acinetobacter tandoii, Acinetobactertjernbergiae, Acinetobacter towneri, or Acinetobacter ursingii have alsobeen linked to infection. Of particular note is the prevalence ofAcinetobacter baumannii infections in US serviceman stationed in theMiddle East, e.g. Iraq. Of concern is the fact that many Acinetobacterstrains appear to be multidrug resistant, thus making the combat ofAcinetobacter infections and contamination difficult. As such, there isan urgent need for safe and effective treatments for Acinetobacterinfections and contamination.

Klebsiella is a genus of non-motile, gram-negative, rod shaped bacteriaKlebsiella species are ubiquitous in nature. In humans, they maycolonize the skin, pharynx, and gastrointestinal tract and may beregarded as normal flora in many parts of the colon, the intestinaltract and in the biliary tract.

Klebsiella species include, Klebsiella granulomatis, Klebsiella oxytoca,Klebsiella pneumoniae, Klebsiella singaporensis, and Klebsiellavariicola, although K. pneumoniae and K. oxytoca are the members of thisgenus responsible for most human infections. Such infections includepneumonia, bacteraemia, thrombophlebitis, urinary tract infection,cholecystitis, diarrhoea, upper respiratory tract infection, woundinfection, osteomyelitis, and meningitis. Rhinoscleroma and ozena aretwo other infections caused by Klebsiella species. Rhinoscleroma is achronic inflammatory process involving the nasopharynx, whereas ozena isa chronic atrophic rhinitis characterized by necrosis of nasal mucosaand mucopurulent nasal discharge.

Klebsiellae often contribute to nosocomial infections. Common sitesinclude the urinary tract, lower respiratory tract, biliary tract, andwounds. The presence of invasive devices, in particular respiratorysupport equipment and urinary catheters, increase the likelihood ofnosocomial infection with Klebsiella species. Sepsis and septic shockmay follow entry of organisms into the blood from these sources.

K. pneumoniae is an important cause of community-acquired pneumonia inelderly persons and subjects with impaired respiratory host defences. K.oxytoca has been implicated in neonatal bacteraemia, especially amongpremature infants and in neonatal intensive care units. Increasingly,the organism is being isolated from patients with neonatal septicaemia.

Problematically, resistance of Klebsiella species to antibiotics isincreasing. As such, there is an urgent need for safe and effectivetreatments for Klebsiella infections and contamination and, inparticular, treatments that overcome antibiotic resistance in Klebsiellaspecies.

Antibiotics are a key tool in the clinical management of bacterialinfections, e.g. those involving the genera mentioned above.Unfortunately, the number of antibiotics available to physicians isfinite and has remained largely unchanged for many years. Resistance ofa bacterium to an antibiotic reduces the number of antibiotics availableto treat the bacterium. Bacteria resistant to multiple antibiotics aretherefore proportionately more difficult to treat. Continued use ofantibiotics inevitably selects for MDR bacteria and so there is anurgent need for techniques by which MDR phenotypes can be overcome. Theinventors have surprisingly found that alginate oligomers can achievethis. Alginates are linear polymers of (1-4) linked β-D-mannuronic acid(M) and/or its C-5 epimer α-L-guluronic acid (G). The primary structureof alginates can vary greatly. The M and G residues can be organised ashomopolymeric blocks of contiguous M or G residues, as blocks ofalternating M and G residues and single M or G residues can be foundinterspacing these block structures. An alginate molecule can comprisesome or all of these structures and such structures might not beuniformly distributed throughout the polymer. In the extreme, thereexists a homopolymer of guluronic acid (polyguluronate) or a homopolymerof mannuronic acid (polymannuronate).

Alginates have been isolated from marine brown algae (e.g. certainspecies of Durvillea, Lessonia and Laminaria) and bacteria such asPseudomonas aeruginosa and Azotobacter vinelandii. Other pseudomonads(e.g. Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonasmendocina) retain the genetic capacity to produce alginates but in thewild they do not produce detectable levels of alginate. By mutationthese non-producing pseudomonads can be induced to produce stably largequantities of alginate.

Alginate is synthesised as polymannuronate and G residues are formed bythe action of epimerases (specifically C-5 epimerases) on the M residuesin the polymer. In the case of alginates extracted from algae, the Gresidues are predominantly organised as G blocks because the enzymesinvolved in alginate biosynthesis in algae preferentially introduce theG neighbouring another G, thus converting stretches of M residues intoG-blocks. Elucidation of these biosynthetic systems has allowed theproduction of alginates with specific primary structures (WO 94/09124,Gimmestad, M et al, Journal of Bacteriology, 2003, Vol 185(12) 3515-3523and WO 2004/011628).

Alginates are typically isolated from natural sources as large highmolecular weight polymers (e.g. an average molecular weight in the range300,000 to 500,000 Daltons). It is known, however, that such largealginate polymers may be degraded, or broken down, e.g. by chemical orenzymatic hydrolysis to produce alginate structures of lower molecularweight. Alginates that are used industrially typically have an averagemolecular weight in the range of 100,000 to 300,000 Daltons (suchalginates are still considered to be large polymers) although alginatesof an average molecular weight of approximately 35,000 Daltons have beenused in pharmaceuticals.

It has now been found that alginate oligomers can be used to overcomeantibiotic resistance and render bacteria that are MDR (resistant tomultiple classes of antibiotics) susceptible to antibiotics (morespecifically susceptible to antibiotic(s) to which they are resistant)and so the use of alginate oligomers together with antibioticsconstitutes a highly effective approach to the combat of contaminationand infections caused by MDR bacteria.

Accordingly, in a first aspect the invention provides a method ofovercoming resistance to at least one antibiotic in an MDR bacterium,said method comprising contacting said bacterium with an alginateoligomer together with (or in conjunction or combination with) theantibiotic.

More particularly, the contacting step may comprise contacting thebacterium (more particularly the bacteria) with an alginate oligomer atthe same, or substantially the same, time or prior to contacting thebacterium with the antibiotic in an amount effective to overcome theresistance of the bacteria to the antibiotic. In particular, the step ofcontacting the bacterium with the alginate oligomer may includeadministering the alginate oligomer to a subject, and in particular to asubject in need of such treatment (e.g. a subject infected with,suspected to be infected with, or at risk of infection with, an MDRbacterium).

Thus the invention provides an alginate oligomer for use together with(or in combination or conjunction with) at least one antibiotic intreating a subject infected, suspected to be infected, or at risk ofinfection, with an MDR bacterium to overcome resistance to theantibiotic in said MDR bacterium.

This aspect of the invention also provides a method of treating asubject infected, suspected to be infected, or at risk of infection,with an MDR bacterium to overcome resistance to the antibiotic in saidMDR bacterium, said method comprising administering an effective amountof the antibiotic to said subject together with an effective amount ofsaid alginate oligomer.

By “use together” it is particularly meant that a pharmaceuticallyeffective amount of the alginate oligomer and a pharmaceuticallyeffective amount of the antibiotic are administered in a manner thatresults in the bacterium (more particularly the bacteria) beingcontacted with an alginate oligomer at the same, or substantially thesame, time or prior to being contacted with the antibiotic. Anyclinically acceptable dosing regime may be used to achieve this. Theskilled man would be able to take into account any relevant variablefactors (e.g. the routes of administration, the bioavailability, and thepharmacokinetics of the oligomer and the antibiotic being used, thesubject's physical state, the location of the bacterium, etc.) in orderto design an appropriate dosing regime for a particular subject. In oneembodiment, a pharmaceutically effective amount of the alginate oligomeris administered at the same or substantially the same time as or priorto administering a pharmaceutically effective amount of the antibiotic.In other embodiments the oligomer is administered separately to andafter the antibiotic. The skilled man would readily be able to designhis dosing regime to maximise the effect of the alginate oligomer andantibiotic he is using in overcoming the resistance of the target MDRbacterium to the antibiotic. He would also be able to select optimalcombinations of the two active agents depending on the particularclinical situation he is faced with. “Use together” does not imply thatthe respective agents are present in the same formulation orcomposition, and accordingly even if used, or administered, at the sameor substantially the same time, the alginate oligomer and antibioticneed not, indeed most likely will not, be present in the samecomposition or formulation, but may be administered separately. Thus“separate” use/administration includes use/administration at the same orsubstantially the same time, or at different times, e.g. sequentially,or at different time intervals according to the desired dosage or usageregime.

The term “infected with” (or “infected by”) is used broadly herein toindicate that the subject may comprise, or contain, or carry, thebacterium in question, i.e. that the bacterium may simply be present inor on the subject, and this may include any site or location in or onthe body of the subject. It is not necessary that the infection of thesubject be manifest as a clinical disease (i.e. that the infectionresult in clinical symptoms in the subject), although this is of courseencompassed. A subject who is suspected to be infected or who is at riskof infection may be a subject who has been exposed to the bacterium orto an infected subject, or a subject presenting with clinical signs orsymptoms of infection (in the case of a suspected infection), or asubject who is susceptible to infection, whether generally e.g. due tothe clinical status of the subject) or particularly to the bacterium inquestion.

Alternatively put, the invention provides the use of an alginateoligomer for the manufacture of a medicament for use together with atleast one antibiotic in treating a subject infected, suspected to beinfected, or at risk of infection, with an MDR bacterium to overcomeresistance to the antibiotic in said MDR bacterium.

The medicament may further comprise the antibiotic (or antibiotics). Themedicament may be in the form of a single composition or formulationcomprising the alginate oligomer and antibiotic(s) or separatecompositions or formulations may be prepared and used, each containingthe alginate oligomer or the antibiotic(s), respectively.

Thus in a more particular aspect the present invention provides the useof an alginate oligomer and at least one antibiotic for the manufactureof a medicament for use in treating a subject infected, suspected to beinfected, or at risk of infection, with an MDR bacterium to overcomeresistance to the antibiotic in said MDR bacterium.

As noted above, the antibiotic may be applied or administered separatelyfrom the alginate oligomer.

Thus a further aspect of the present invention provides a productcontaining an alginate oligomer and an antibiotic (e.g. one or moreantibiotics) as a combined preparation for separate, simultaneous orsequential use, in treating a subject infected, suspected to beinfected, or at risk of infection, with an MDR bacterium to overcomeresistance to the antibiotic in said MDR bacterium.

The antibiotic may be applied or administered simultaneously with thealginate oligomer or sequentially. As noted above, in one embodiment theantibiotic is administered at the same or substantially the same time asthe alginate oligomer, and in another embodiment it is administeredafter the alginate oligomer. In other embodiments the oligomer isadministered separately to and after the antibiotic. Included within thescope of “substantially the same time” is application or administrationof the antibiotic immediately or almost immediately before or after thealginate oligomer. The term “almost immediately” may be read asincluding application or administration within one hour of the previousapplication or administration, preferably within 30 minutes. However theantibiotic may be applied or administered at least 1 hour, at least 3hours, or at least 6 hours or more after the alginate oligomer. In theseembodiments the antibiotic can be applied or administered with orwithout a further application of an alginate oligomer. The alginateoligomer can be applied or administered in a plurality of applicationsprior to or with the antibiotic, including as noted above, anapplication or administration immediately or almost immediately afterthe antibiotic. In other embodiments the antibiotic(s) may convenientlybe applied or administered before the alginate oligomer, e.g. at least 1hour, at least 3 hours, at least 6 hours before the alginate oligomer.In these embodiments the alginate oligomer can be applied oradministered with or without a further application of the antibiotic.The antibiotic can be applied or administered in a plurality ofapplications prior to or with the alginate oligomer.

As noted above, alginates typically occur as polymers of an averagemolecular weight of at least 35,000 Daltons i.e. approximately 175 toapproximately 190 monomer residues, although typically much higher andan alginate oligomer according to the present invention may be definedas a material obtained by fractionation (i.e. size reduction) of analginate polymer, commonly a naturally occurring alginate. An alginateoligomer can be considered to be an alginate of an average molecularweight of less than 35,000 Daltons (i.e. less than approximately 190 orless than approximately 175 monomer residues), in particular an alginateof an average molecular weight of less than 30,000 Daltons (i.e. lessthan approximately 175 or less than approximately 150 monomer residues)more particularly an average molecular weight of less than 25,000 or20,000 Daltons (i.e. less than approximately 135 or 125 monomer residuesor less than approximately 110 or 100 monomer residues).

Viewed alternatively, an oligomer generally comprises 2 or more units orresidues and an alginate oligomer for use according to the inventionwill typically contain 2 to 100 monomer residues, preferably 2 to 75,preferably 2 to 50, more preferably 2 to 40, 2 to 35 or 2 to 30residues. Thus an alginate oligomer for use according to the inventionwill typically have an average molecular weight of 350 to 20,000Daltons, preferably 350 to 15,000 Daltons, preferably 350 to 10,000Daltons and more preferably 350 to 8000 Daltons, 350 to 7000 Daltons, or350 to 6,000 Daltons.

Alternatively put, the alginate oligomer may have a degree ofpolymerisation (DP), or a number average degree of polymerisation (DPn)of 2 to 100, preferably 2 to 75, preferably 2 to 50, more preferably 2to 40, 2 to 35, 2 to 30, 2 to 28, 2 to 25, 2 to 22, 2 to 20, 2 to 18, 2to 17, 2 to 15 or 2 to 12.

Other representative ranges (whether for the number of residues, DP orDPn) include any one of 3, 4, 5, 6, 7, 8, 9, 10 or 11 to any one of 50,45, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24,23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12.

Other representative ranges (whether for the number of residues, DP orDPn) include any one of 8, 9, 10, 11, 12, 13, 14 or 15 to any one of 50,45, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24,23, 22, 21, 20, 19, 18, 17 or 16.

Other representative ranges (whether for the number of residues, DP orDPn) include any one of 11, 12, 13, 14, 15, 16, 17 or 18 to any one of50, 45, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25,24, 23, 22, 21, 20 or 19.

An alginate oligomer will, as noted above, contain (or comprise)guluronate or guluronic acid (G) and/or mannuronate or mannuronic acid(M) residues or units. An alginate oligomer according to the inventionwill preferably be composed solely, or substantially solely (i.e.consist essentially of) uronate/uronic acid residues, more particularlysolely or substantially solely of G and/or M residues. Alternativelyexpressed, in the alginate oligomer of use in the present invention, atleast 80%, more particularly at least 85, 90, 95 or 99% of the monomerresidues may be uronate/uronic acid residues, or, more particularly Gand/or M residues. In other words, preferably the alginate oligomer willnot comprise other residues or units (e.g. other saccharide residues, ormore particularly other uronic acid/uronate residues).

The alginate oligomer is preferably a linear oligomer.

More particularly, in a preferred embodiment at least 30% of the monomerresidues of the alginate oligomer are G residues (i.e. guluronate orguluronic acid). In other words the alginate oligomer will contain atleast 30% guluronate (or guluronic acid) residues. Specific embodimentsthus include alginate oligomers with (e.g. containing) 30 to 70% G(guluronate) residues or 70 to 100% G (guluronate) residues. Thus, arepresentative alginate oligomer for use according to the presentinvention may contain at least 70% G residues (i.e. at least 70% of themonomer residues of the alginate oligomer will be G residues).

Preferably at least 50% or 60%, more particularly at least 70% or 75%,even more particularly at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97,98 or 99% of the monomer residues are guluronate. In one embodiment thealginate oligomer may be an oligoguluronate (i.e. a homooligomer of G,or 100% G)

In a further preferred embodiment, the above described alginates of theinvention have a primary structure wherein the majority of the Gresidues are in so called G-blocks. Preferably at least 50%, morepreferably at least 70 or 75%, and most preferably at least 80, 85, 90,92 or 95% of the G residues are in G-blocks. A G block is a contiguoussequence of at least two G residues, preferably at least 3 contiguous Gresidues, more preferably at least 4 or 5 contiguous G residues, mostpreferably at least 7 contiguous G residues.

In particular at least 90% of the G residues are linked 1-4 to another Gresidue. More particularly at least 95%, more preferably at least 98%,and most preferably at least 99% of the G residues of the alginate arelinked 1-4 to another G residue.

The alginate oligomer of use in the invention is preferably a 3- to35-mer, more preferably a 3- to 28-mer, in particular a 4- to 25-mer,especially a 6- to 22-mer, in particular an 8- to 20-mer, especially a10- to 15-mer, e.g. having a molecular weight in the range 350 to 6400Daltons or 350 to 6000 Daltons, preferably 550 to 5500 Daltons,preferably 750 to 5000 Daltons, and especially 750 to 4500 Daltons or2000 to 3000 Daltons. Other representative alginate oligomers include,as mentioned above, oligomers with 7, 8, 9, 10, 11 or 12 to 50, 45, 40,35, 28, 25, 22 or 20 residues.

It may be a single compound or it may be a mixture of compounds, e.g. ofa range of degrees of polymerization. As noted above, the monomericresidues in the alginate oligomer, may be the same or different and notall need carry electrically charged groups although it is preferred thatthe majority (e.g. at least 60%, preferably at least 80% more preferablyat least 90%) do. It is preferred that a substantial majority, e.g. atleast 80%, more preferably at least 90% of the charged groups have thesame polarity. In the alginate oligomer, the ratio of hydroxyl groups tocharged groups is preferably at least 2:1, more especially at least 3:1.

The alginate oligomer of the invention may have a degree ofpolymerisation (DP), or a number average degree of polymerisation(DP_(n)), of 3-28, 4-25, 6-22, 8-20 or 10-15, or 5 to 18 or 7 to 15 or 8to 12, especially 10.

The alginate oligomer of the invention may have a degree ofpolymerisation (DP), or a number average degree of polymerisation(DP_(n)), of 8-50, 8-40, 8-35, 8-30, 8-28, 8-25, 8-22, 8-20, 8-18, 8-16or 8-14.

The alginate oligomer of the invention may have a degree ofpolymerisation (DP), or a number average degree of polymerisation(DP_(n)), of 9-50, 9-40, 9-35, 9-30, 9-28, 9-25, 9-22, 9-20, 9-18, 9-16or 9-14.

The alginate oligomer of the invention may have a degree ofpolymerisation (DP), or a number average degree of polymerisation(DP_(n)), of 10-50, 10-40, 10-35, 10-30, 10-28, 10-25, 10-22, 10-20,10-18, 10-16 or 10-14.

The alginate oligomer of the invention may have a degree ofpolymerisation (DP), or a number average degree of polymerisation(DP_(n)), of 12-50, 12-40, 12-35, 12-30, 12-28, 12-25, 12-22, 12-20,12-18, 12-16 or 12-14.

The alginate oligomer of the invention may have a degree ofpolymerisation (DP), or a number average degree of polymerisation(DP_(n)), of 15-50, 15-40, 15-35, 15-30, 15-28, 15-25, 15-22, 15-20,15-18 or 15-16.

The alginate oligomer of the invention may have a degree ofpolymerisation (DP), or a number average degree of polymerisation(DP_(n)), of 18-50, 18-40, 18-35, 18-30, 18-28, 18-25, 18-22 or 18-20.

Preferably the alginate oligomer of the invention is substantially free,preferably essentially free, of alginate oligomers having a degree ofpolymerisation outside of the ranges disclosed herein. This may beexpressed in terms of the molecular weight distribution of the alginateoligomer of the invention, e.g. the percentage of each mole of thealginate oligomer being used in accordance with the invention which hasa DP outside the relevant range. The molecular weight distribution ispreferably such that no more than 10%, preferably no more than 9, 8, 7,6, 5, 4, 3, 2, or 1% mole has a DP of three, two or one higher than therelevant upper limit for DP_(n). Likewise it is preferred that no morethan 10%, preferably no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1% mole hasa DP below a number three, two or one smaller than the relevant lowerlimit for DP_(n).

Suitable alginate oligomers are described in WO2007/039754,WO2007/039760, WO 2008/125828, and WO2009/068841, the disclosures ofwhich are explicitly incorporated by reference herein in their entirety.

Representative suitable alginate oligomers have a DP_(n) in the range 5to 30, a guluronate/galacturonate fraction (F_(G)) of at least 0.80, amannuronate fraction (F_(M)) of no more than 0.20, and at least 95 mole% of DP no more than 25.

Further suitable alginate oligomers have a number average degree ofpolymerization in the range 7 to 15 (preferably 8 to 12), aguluronate/galacturonate fraction (F_(G)) of at least 0.85 (preferablyat least 0.90), a mannuronate fraction (F_(M)) of no more than 0.15(preferably no more than 0.10), and having at least 95% mole with adegree of polymerization less than 17 (preferably less than 14).

Further suitable alginate oligomers have a number average degree ofpolymerization in the range 5 to 18 (especially 7 to 15), aguluronate/galacturonate fraction (F_(G)) of at least 0.80 (preferablyat least 0.85, especially at least 0.92), a mannuronate fraction (F_(M))of no more than 0.20 (preferably no more than 0.15, especially no morethan 0.08), and having at least 95% mole with a degree of polymerizationless than 20 (preferably less than 17).

Further suitable alginate oligomers have a number average degree ofpolymerization in the range 5 to 18, a guluronate/galacturonate fraction(F_(G)) of at least 0.92, a mannuronate fraction (F_(M)) of no more than0.08, and having at least 95% mole with a degree of polymerization lessthan 20.

Further suitable alginate oligomers have a number average degree ofpolymerization in the range 5 to 18 (preferably 7 to 15, more preferably8 to 12, especially about 10), a guluronate/galacturonate fraction(F_(G)) of at least 0.80 (preferably at least 0.85, more preferably atleast 0.90, especially at least 0.92, most especially at least 0.95), amannuronate fraction (F_(M)) of no more than 0.20 (preferably no morethan 0.15, more preferably no more than 0.10, especially no more than0.08, most especially no more than 0.05), and having at least 95% molewith a degree of polymerization less than 20 (preferably less than 17,more preferably less than 14).

Further suitable alginate oligomers have a number average degree ofpolymerization in the range 7 to 15 (preferably 8 to 12), aguluronate/galacturonate fraction (F_(G)) of at least 0.92 (preferablyat least 0.95), a mannuronate fraction (F_(M)) of no more than 0.08(preferably no more than 0.05), and having at least 95% mole with adegree of polymerization less than 17 (preferably less than 14).

Further suitable alginate oligomers have a number average degree ofpolymerization in the range 5 to 18, a guluronate/galacturonate fraction(F_(G)) of at least 0.80, a mannuronate fraction (F_(M)) of no more than0.20, and having at least 95% mole with a degree of polymerization lessthan 20.

Further suitable alginate oligomers have a number average degree ofpolymerization in the range 7 to 15, a guluronate/galacturonate fraction(F_(G)) of at least 0.85, a mannuronate fraction (F_(M)) of no more than0.15, and having at least 95% mole with a degree of polymerization lessthan 17.

Further suitable alginate oligomers have a number average degree ofpolymerization in the range 7 to 15, a guluronate/galacturonate fraction(F_(G)) of at least 0.92, a mannuronate fraction (F_(M)) of no more than0.08, and having at least 95% mole with a degree of polymerization lessthan 17.

It will thus be seen that a particular class of alginate oligomersfavoured according to the present invention is alginate oligomersdefined as so-called “high G” or “G-block” oligomers i.e. having a highcontent of G residues or G-blocks (e.g. wherein at least 70% of themonomer residues are G, preferably arranged in G-blocks). However, othertypes of alginate oligomer may also be used, including in particular“high M” or “M-block” oligomers or MG-block oligomers, as describedfurther below. Accordingly, it is alginate oligomers with highproportions of a single monomer type, and with said monomers of thistype being present predominantly in contiguous sequences of that monomertype, that represent oligomers that are particularly preferred, e.g.oligomers wherein at least 70% of the monomer residues in the oligomerare G residues linked 1-4 to another G-residue, or more preferably atleast 75%, and most preferably at least 80, 85, 90, 92, 93, 94, 95, 96,97, 98, 99% of the monomers residues of the oligomer are G residueslinked 1-4 to another G residue. This 1-4 linkage of two G residues canbe alternatively expressed as a guluronic unit bound to an adjacentguluronic unit.

In a further embodiment at least, or more particularly more than, 50% ofthe monomer residues of the alginate oligomer may be M residues (i.e.mannuronate or mannuronic acid). In other words the alginate oligomerwill contain at least or alternatively more than 50% mannuronate (ormannuronic acid) residues. Specific embodiments thus include alginateoligomers with (e.g. containing) 50 to 70% M (mannuronate) residues ore.g. 70 to 100% M (mannuronate) residues. Further specific embodimentsalso include oligomers containing 71 to 85% M residues or 85 to 100% Mresidues. Thus, a representative alginate oligomer for use according tothis embodiment of the present invention will contain more than 70% Mresidues (i.e. more than 70% of the monomer residues of the alginateoligomer will be M residues).

In other embodiments at least 50% or 60%, more particularly at least 70%or 75%, even more particularly at least 80, 85, 90, 95 or 99% of themonomer residues are mannuronate. In one embodiment the alginateoligomer may be an oligomannuronate (i.e. a homooligomer of M, or 100%M).

In a further embodiment, the above described alginates of the inventionhave a primary structure wherein the majority of the M residues are inso called M-blocks. In this embodiment preferably at least 50%, morepreferably at least 70 or 75%, and most preferably at least 80, 85, 90or 95% of the M residues are in M-blocks. An M block is a contiguoussequence of at least two M residues, preferably at least 3 contiguous Mresidues, more preferably at least 4 or 5 contiguous M residues, mostpreferably at least 7 contiguous M residues.

In particular, at least 90% of the M residues are linked 1-4 to anotherM residue. More particularly at least 95%, more preferably at least 98%,and most preferably at least 99% of the M residues of the alginate arelinked 1-4 to another M residue.

Other preferred oligomers are alginate oligomers wherein at least 70% ofthe monomer residues in the oligomer are M residues linked 1-4 toanother M-residue, or more preferably at least 75%, and most preferablyat least 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99% of the monomersresidues of the oligomer are M residues linked 1-4 to another M residue.This 1-4 linkage of two M residues can be alternatively expressed as amannuronic unit bound to an adjacent mannuronic unit.

In a still further embodiment, the alginate oligomers of the inventioncomprise a sequence of alternating M and G residues. A sequence of atleast three, preferably at least four, alternating M and G residuesrepresents an MG block. Preferably the alginate oligomers of theinvention comprise an MG block. Expressed more specifically, an MG blockis a sequence of at least three contiguous residues consisting of G andM residues and wherein each non-terminal (internal) G residue in thecontiguous sequence is linked 1-4 and 4-1 to an M residue and eachnon-terminal (internal) M residue in the contiguous sequence is linked1-4 and 4-1 to a G residue. Preferably the MG block is at least 5 or 6contiguous residues, more preferably at least 7 or 8 contiguousresidues.

In a further embodiment the minority uronate in the alginate oligomer(i.e. mannuronate or guluronate) is found predominantly in MG blocks. Inthis embodiment preferably at least 50%, more preferably at least 70 or75% and most preferably at least 80, 85, 90 or 95% of the minorityuronate monomers in the MG block alginate oligomer are present in MGblocks. In another embodiment the alginate oligomer is arranged suchthat at least 50%, at least 60%, at least 70%, at least 80%, at least85%, at least 90%, at least 95%, at least 99%, e.g. 100% of the G and Mresidues in the oligomer are arranged in MG blocks.

Although at its broadest, the invention extends to embodiments whereinat least 1% but less than 100% of the monomer residues of the oligomerare G residues (i.e. guluronate or guluronic acid), more particularly,and as defined further below, at least 30% of the monomer residues are Gresidues. Thus, at its broadest the MG block containing alginateoligomer may contain at least 1%, but less than 100%, guluronate (orguluronic acid) residues, but generally the MG block containing alginateoligomer will contain at least 30% (or at least 35, 40 or 45% or 50% G)but less than 100% G. Specific embodiments thus include MG blockcontaining alginate oligomers with (e.g. containing) 1 to 30% G(guluronate) residues, 30 to 70% G (guluronate) residues or 70 to 99% G(guluronate) residues. Thus, a representative MG block containingalginate oligomer for use according to the present invention may containmore than 30%, but less than 70%, G residues (i.e. more than 30%, butless than 70%, of the monomer residues of the MG block alginate oligomerwill be G residues).

Preferably more than 30%, more particularly more than 35% or 40%, evenmore particularly more than 45, 50, 55, 60 or 65%, but in each, caseless than 70%, of the monomer residues of the MG block containingalginate oligomer are guluronate. Alternatively, less than 70%, morepreferably less than 65% or 60%, even more preferably less than 55, 50,45, 40 or 35%, but in each case more than 30% of the monomer residues ofthe MG block containing alginate oligomer are guluronate. Any rangeformed by any combination of these values may be chosen. Therefore forinstance the MG block containing alginate oligomer can have e.g. between35% and 65%, 40% and 60% or 45% and 55% G residues.

In another embodiment the MG block containing alginate oligomer may haveapproximately equal amounts of G and M residues (e.g. ratios between 65%G/35% M and 35% G/65% M, for instance 60% G/40% M and 40% G/60% M; 55%G/45% M and 45% G/55% M; 53% G/47% M and 47% G/53% M; 51% G/49% M and49% G/51% M; e.g. about 50% G and about 50% M) and these residues arearranged predominantly, preferably entirely or as completely aspossible, in an alternating MG pattern (e.g. at least 50% or at least60, 70, 80, 85, 90 or 95% or 100% of the M and G residues are in analternating MG sequence).

In certain embodiments the terminal uronic acid residues of theoligomers of the invention do not have a double bond, especially adouble bond situated between the C₄ and C₅ atom. Such oligomers may bedescribed as having saturated terminal uronic acid residues. The skilledman would be able to prepare oligomers with saturated terminal uronicacid residues without undue burden. This may be through the use ofproduction techniques which yield such oligomers, or by converting(saturating) oligomers produced by processes that yield oligomers withunsaturated terminal uronic acid residues.

The alginate oligomer will typically carry a charge and so counter ionsfor the alginate oligomer may be any physiologically tolerable ion,especially those commonly used for charged drug substances, e.g. sodium,potassium, ammonium, chloride, mesylate, meglumine, etc. Ions whichpromote alginate gelation e.g. group 2 metal ions may also be used.

While the alginate oligomer may be a synthetic material generated fromthe polymerisation of appropriate numbers of guluronate and mannuronateresidues, the alginate oligomers of use in the invention mayconveniently be obtained, produced or derived from natural sources suchas those mentioned above, namely natural alginate source materials.

Polysaccharide to oligosaccharide cleavage to produce the alginateoligomer useable according to the present invention may be performedusing conventional polysaccharide lysis techniques such as enzymaticdigestion and acid hydrolysis. In one favoured embodiment acidhydrolysis is used to prepare the alginate oligomers on the invention.In other embodiments enzymic digestion is used with an additionalprocessing step(s) to saturate the terminal uronic acids in theoligomers.

Oligomers may then be separated from the polysaccharide breakdownproducts chromatographically using an ion exchange resin or byfractionated precipitation or solubilisation or filtration. U.S. Pat.No. 6,121,441 and WO 2008/125828, which are explicitly incorporated byreference herein in their entirety, describe a process suitable forpreparing the alginate oligomers of use in the invention. Furtherinformation and discussion can be found in for example in “Handbooks ofHydrocolloids”, Ed. Phillips and Williams, CRC, Boca Raton, Fla., USA,2000, which textbook is explicitly incorporated by reference herein inits entirety.

The alginate oligomers may also be chemically modified, including butnot limited to modification to add charged groups (such as carboxylatedor carboxymethylated glycans) and alginate oligomers modified to alterflexibility (e.g. by periodate oxidation).

Alginate oligomers (for example oligoguluronic acids) suitable for useaccording to the invention may conveniently be produced by acidhydrolysis of alginic acid from, but not limited to, Laminaria hyperboraand Lessonia nigrescens, dissolution at neutral pH, addition of mineralacid reduce the pH to 3.4 to precipitate the alginate oligomer(oligoguluronic acid), washing with weak acid, resuspension at neutralpH and freeze drying.

The alginates for production of alginate oligomers of the invention canalso be obtained directly from suitable bacterial sources e.g.Pseudomonas aeruginosa or Azotobacter vinelandii.

In embodiments where alginate oligomers which have primary structures inwhich the majority of the G residues are arranged in G-blocks ratherthan as single residues are required, algal sources are expected to bemost suitable on account of the fact that the alginates produced inthese organisms tend to have these structures. The bacterial sources maybe more suitable for obtaining alginate oligomers of differentstructures.

The molecular apparatus involved in alginate biosynthesis in Pseudomonasfluorescens and Azotobacter vinelandii has been cloned and characterised(WO 94/09124; Ertesvag, H., et al, Metabolic Engineering, 1999, Vol 1,262-269; WO 2004/011628; Gimmestad, M., et al (supra); Remminghorst andRehm, Biotechnology Letters, 2006, Vol 28, 1701-1712; Gimmestad, M. etal, Journal of Bacteriology, 2006, Vol 188(15), 5551-5560) and alginatesof tailored primary structures can be readily obtained by manipulatingthese systems.

The G content of alginates (for example an algal source material) can beincreased by epimerisation, for example with mannuronan C-5 epimerasesfrom A. vinelandii or other epimerase enzymes. Thus, for example invitro epimerisation may be carried out with isolated epimerases fromPseudomonas or Azotobacter, e.g. AlgG from Pseudomonas fluorescens orAzotobacter vinelandii or the AlgE enzymes (AlgE1 to AlgE7) fromAzotobacter vinelandii. The use of epimerases from other organisms thathave the capability of producing alginate, particularly algae, is alsospecifically contemplated. The in vitro epimerisation of low G alginateswith Azotobacter vinelandii AlgE epimerases is described in detail inErtesvag et al (supra) and Strugala et al (Gums and Stabilisers for theFood Industry, 2004, 12, The Royal Society of Chemistry, 84-94).

To obtain G-block containing alginates or alginate oligomers,epimerisation with one or more Azotobacter vinelandii AlgE epimerasesother than AlgE₄ is preferred as these enzymes are capable of producingG block structures. On the other hand AlgE4 epimerase can be used tocreate alginates or alginate oligomers with alternating stretches of M/Gsequence or primary structures containing single G residue as it hasbeen found that this enzyme seems preferentially to epimerise individualM residues so as to produce single G residues linked to M residuesrather than producing G blocks. Particular primary structures can beobtained by using different combinations of these enzymes.

Mutated versions of these enzymes or homologues from other organisms arealso specifically contemplated as of use. WO 94/09124 describesrecombinant or modified mannuronan C-5 epimerase enzymes (AlgE enzymes)for example encoded by epimerase sequences in which the DNA sequencesencoding the different domains or modules of the epimerases have beenshuffled or deleted and recombined. Alternatively, mutants of naturallyoccurring epimerase enzymes, (AlgG or AlgE) may be used, obtained forexample by site directed or random mutagenesis of the AlgG or AlgEgenes.

A different approach is to create Pseudomonas and Azotobacter organismsthat are mutated in some or all of their epimerase genes in such a waythat those mutants produce alginates of the required structure forsubsequent alginate oligomer production, or even alginate oligomers ofthe required structure and size (or molecular weight). The generation ofa number of Pseudomonas fluorescens organisms with mutated AlgG genes isdescribed in detail in WO 2004/011628 and Gimmestad, M., et al, 2003(supra). The generation of a number of Azotobacter vinelandii organismswith mutated AlgE genes is disclosed in Gimmestad, M., et al, 2006(supra). The skilled man would be able to use this teaching to producenew mutants that could be used to give rise to the alginate oligomers ofthe invention without undue burden.

A further approach is to delete or inactivate the endogenous epimerasegenes from an Azotobacter or a Pseudomonas organism and then tointroduce one or more exogenous epimerase genes, which may or may not bemutated (i.e. may be wild-type or modified) and the expression of whichmay be controlled, for example by the use of inducible or other“controllable promoters”. By selecting appropriate combinations ofgenes, alginates of predetermined primary structure can be produced.

A still further approach would be to introduce some or all of thealginate biosynthesis machinery of Pseudomonas and/or Azotobacter into anon-alginate producing organism (e.g. E. coli) and to induce theproduction of alginate from these genetically modified organisms.

When these culture-based systems are used, the primary structure of thealginate or alginate oligomer products can be influenced by the cultureconditions. It is well within the capabilities of the skilled man toadjust culture parameters such as temperature, osmolarity, nutrientlevels/sources and atmospheric parameters in order to manipulate theprimary structure of the alginates produced by a particular organism.

References to “G residues/G” and “M residues/M” or to guluronic acid ormannuronic acid, or guluronate or mannuronate are to be readinterchangeably as references to guluronic acid/guluronate andmannuronic acid/mannuronate (specifically α-L-guluronic acid/guluronateand β-D-mannuronic acid/mannuronate), and further include derivativesthereof in which one or more available side chains or groups have beenmodified without resulting in a capacity to overcome antibioticresistance that is substantially lower than that of the unmodifiedoligomer. Common saccharide modifying groups would include acetyl,sulphate, amino, deoxy, alcohol, aldehyde, ketone, ester and anhydrogroups. The alginate oligomers may also be chemically modified to addcharged groups (such as carboxylated or carboxymethylated glycans), andto alter flexibility (e.g. by periodate oxidation). The skilled manwould be aware of still further chemical modifications that can be madeto the monosaccharide subunits of oligosaccharides and these can beapplied to the alginate oligomers of the invention.

The bacterium targeted by the method of the invention can be anybacterium that is MDR, which according to the present invention meansthat the bacterium is resistant to at least 3, or at least 4, 5, 6, 7,8, 9 or 10 antibiotic classes. As noted above antibiotics in differentclasses are structurally and/or functionally different. In otherembodiments the bacterium targeted by the method of the invention can beany bacterium that has extreme drug resistance, which according to thepresent invention means that the bacterium is resistant to the majorityof, or all, antibiotics. In particular, extreme drug resistant bacteriumare resistant to at least one antibiotic of last resort (e.g.vancomycin, linezolid, etc.). The skilled man would be aware of examplesof antibiotics of last resort

Classes of antibiotics and representative constituents thereof include,but are not limited to the aminoglycosides (e.g. amikacin, gentamicin,kanamycin, neomycin, netilmicin, streptomycin, tobramycin); thecarbacephems (e.g. loracarbef); the 1st generation cephalosporins (e.g.cefadroxil, cefazolin, cephalexin); 2nd generation cephalosporins (e.g.cefaclor, cefamandole, cephalexin, cefoxitin, cefprozil, cefuroxime);3rd generation cephalosporins (e.g. cefixime, cefdinir, cefditoren,cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten,ceftizoxime, ceftriaxone); 4th generation cephalosporins (e.g.cefepime); the macrolides (e.g. azithromycin, clarithromycin,dirithromycin, erythromycin, troleandomycin); the monobactams (e.g.aztreonam); the penicillins (e.g. amoxicillin, ampicillin,carbenicillin, cloxacillin, dicloxacillin, nafcillin, oxacillin,penicillin G, penicillin V, piperacillin, ticarcillin); the polypeptideantibiotics (e.g. bacitracin, colistin, polymyxin B); the quinolones(e.g. ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin,moxifloxacin, norfloxacin, ofloxacin, trovafloxacin); the sulfonamides(e.g. mafenide, sulfacetamide, sulfamethizole, sulfasalazine,sulfisoxazole, trimethoprim-sulfamethoxazole); the tetracyclines (e.g.demeclocycline, doxycycline, minocycline, oxytetracycline,tetracycline); the glycylcyclines (e.g. tigecycline); the carbapenems(e.g. imipenem, meropenem, ertapenem, doripenem, panipenem/betamipron,biapenem, PZ-601); other antibiotics include chloramphenicol;clindamycin, ethambutol; fosfomycin; isoniazid; linezolid;metronidazole; nitrofurantoin; pyrazinamide; quinupristin/dalfopristin;rifampin; spectinomycin; and vancomycin.

In preferred embodiments of the invention the MDR bacteria are resistantto three or more antibiotic classes selected from the macrolides, theβ-lactams (which may include the carbapenems and/or monobactams and/orcarbacephems), the tetracyclines, the polypeptide antibiotics and thequinolones. In other embodiments, the classes may include theaminoglycosides. In still further embodiments the classes may includethe macrolides, the β-lactams and the quinolones. It will be noted thatinvention may result in the overcoming of resistance to one or moreclasses to which the MDR bacterium is resistant, but it is notnecessarily implied that resistance is overcome to all of the classes ofantibiotic to which an MDR bacterium may be resistant. Thus for exampleresistance to a macrolide and/or a β-lactam and/or a quinolone may beovercome in an MDR strain which is also resistant to other antibioticse.g. aminoglycosides.

More specifically, in these embodiments the antibiotic classes may beselected from the macrolides, the monobactams, the carbapenems, thecarbacephems, the 3rd and 4th generation cephalosporins, thetetracyclines, the polypeptide antibiotics and the quinolones. In moreparticular representative embodiments the bacteria may be resistant tothree or more antibiotic classes selected from macrolides, β-lactams,and quinolones e.g. three or more antibiotic classes selected frommacrolides, monobactams, carbapenems, carbacephems, 3rd and 4thgeneration cephalosporins, and quinolones. In other embodiments, theantibiotic classes listed above may also include the aminoglycosides.For example, the antibiotics may be selected from amikacin, gentamicin,kanamycin, neomycin, netilmicin, streptomycin, tobramycin, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin,telithromycin, CarbomycinA, josamycin, kitasamycin, midecamicine,oleandomycin, spiramycin, tylosin, troleandomycin, aztreonam, imipenem,meropenem, ertapenem, doripenem, panipenem/betamipron, biapenem, PZ-601,cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime,ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime,demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline,bacitracin, colistin, polymyxin B, ciproftoxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,ofloxacin, and/or trovafloxacin. In particular, the MDR bacteria may beresistant to one or more antibiotics selected from amikacin, tobramycin,ceftazidime, imipenem/cilastatin, meropenem, aztreonam, oxytetracycline,colistin, azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, spiramycin and ciprofloxacin, and it is particularlypreferred that the MDR bacteria are resistant to one or more antibioticsselected from ceftazidime, imipenem/cilastatin, meropenem, aztreonam,azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, spiramycin and ciprofloxacin. More preferably the MDRbacteria are resistant to one or more antibiotics selected fromaztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, spiramycin and ciprofloxacin.

In other embodiments the MDR bacteria are at least resistant to anantibiotic class selected from the β-lactams (e.g. the 1st and 2ndgeneration cephalosporins and/or monobactams) and the macrolides. Suchbacteria may also be resistant to aminoglycosides and/or quinolones(e.g. fluoroquinolones). In other embodiments the MDR bacteria are atleast resistant to an antibiotic selected from amikacin, gentamicin,kanamycin, neomycin, netilmicin, streptomycin, tobramycin, cefadroxil,cefazolin, cephalexin, cefaclor, cefamandole, cephalexin, cefoxitin,cefprozil, cefuroxime, azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, telithromycin, CarbomycinA, josamycin,kitasamycin, midecamicine, oleandomycin, spiramycin, troleandomycin andtylosin, or any combination thereof; e.g. amikacin, tobramycin,gentamicin and netilmicin, or any combination thereof.

In a particular embodiment, alginate oligomers may be used according tothe present invention to overcome resistance to azithromycin and/orciprofloxacin, or more generally the antibiotic classes to which theybelong, namely macrolides (e.g. azithromycin, clarithromycin,dirithromycin, erythromycin, roxithromycin, telithromycin, CarbomycinA,josamycin, kitasamycin, midecamicine, oleandomycin, spiramycin,troleandromycin, tylosin) and quinolones (e.g. ciprofloxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,ofloxacin, trovafloxacin).

As shown in the Examples below, alginate oligomers have been found to beparticularly effective in potentiating the effects of these classes ofantibiotics (namely the macrolides and/or quinolones). Additionally,alginate oligomers are particularly effective in potentiating theeffects of β-lactam antibiotics, and may also potentiate the effects ofother antibiotics. In the cases of the three classes of antibioticsmentioned above, namely macrolides, quinolones and/or β-lactams,alginate oligomers can be seen to have a synergistic effect with theantibiotics. More particularly, the potentiating effect of the alginateoligomers may be seen also with bacteria that are not MDR. Accordingly,more broadly viewed, the invention can be seen to relate to the use ofalginate oligomers in conjunction (or combination) with a macrolide,quinolone and/or β-lactam antibiotic, e.g. to combat bacteria, moreparticularly to treat or combat bacterial infection and/or contamination(i.e. colonisation), or alternatively put for example, to potentiate theeffect of the antibiotic. This is discussed in more detail below.

In the context of the “MDR” aspects of the present invention, thealginate oligomers of the invention may be used to overcome resistancein MDR bacteria to one or more of any of the above-mentioned antibioticsand the methods of the invention therefore encompass the use of thealginate oligomers of the invention together with an antibiotic to whichan MDR bacterium is resistant to combat that MDR bacterium. In preferredembodiments of the methods of the invention the antibiotic used is anantibiotic selected from the macrolides, the β-lactams, thetetracyclines, and the quinolones. In a further embodiment thepolypeptide antibiotics and/or the aminoglycosides may be included. Inalternative embodiments the antibiotic does not include anaminoglycoside and/or a polypeptide antibiotic (e.g. colistin). Morespecifically, in the embodiments set out above, the antibiotic may beselected from the macrolides, the monobactams, the carbapenems, thecarbacephems, the 3rd and 4th generation cephalosporins, thetetracyclines, and the quinolones. In more particular representativeembodiments the antibiotic may be selected from macrolides, β-lactams,tetracyclines and quinolones e.g. macrolides, monobactams, carbapenems,carbacephems, 3rd and 4th generation cephalosporins, tetracyclines andquinolones. In more particular representative embodiments the antibioticmay be selected from macrolides, β-lactams and quinolones e.g.macrolides, monobactams, carbapenems, carbacephems, 3rd and 4thgeneration cephalosporins and quinolones. For example, the antibioticmay be selected from amikacin, gentamicin, kanamycin, neomycin,netilmicin, streptomycin, tobramycin, azithromycin, clarithromycin,dirithromycin, erythromycin, roxithromycin, telithromycin, CarbomycinA,josamycin, kitasamycin, midecamicine, oleandomycin, spiramycin, tylosin,troleandomycin, aztreonam, imipenem, meropenem, ertapenem, doripenem,panipenem/betamipron, biapenem, PZ-601, cefixime, cefdinir, cefditoren,cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten,ceftizoxime, ceftriaxone, cefepime, demeclocycline, doxycycline,minocycline, oxytetracycline, tetracycline, bacitracin, colistin,polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin,lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, and trovafloxacin.In particular, the antibiotic may selected from ceftazidime,imipenem/cilastatin, meropenem, aztreonam, oxytetracycline,azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, spiramycin and ciprofloxacin, and it is particularlypreferred that the antibiotic is selected from ceftazidime,imipenem/cilastatin, meropenem, aztreonam, azithromycin, clarithromycin,dirithromycin, erythromycin, roxithromycin, spiramycin andciprofloxacin. More preferably the antibiotic is selected fromaztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, spiramycin and ciprofloxacin. In other embodiments theantibiotic used is not tobramycin, amikacin and/or colistin. In otherembodiments the antibiotic used is not an aminoglycoside or apolypeptide antibiotic. In other embodiments the antibiotic used is notan antibiotic that has a positive charge under the conditions in whichit will be used with the alginate oligomer, e.g. antibiotics with atleast 3, e.g. at least 4, 5, 6 or 7 amino (—NH₂) groups.

As noted above, in more general terms, the alginate oligomers of theinvention are effective in potentiating the effects of antibiotics, e.g.any of those discussed above. The alginate oligomers of the inventionmay thus be used to increase (or improve) the efficacy of antibioticsgenerally. Particularly good effects have been observed with macrolides,β-lactams, tetracyclines and quinolones e.g. macrolides, monobactams,carbapenems, 3rd and 4th generation cephalosporins, tetracyclines andquinolones; and in particular ceftazidime, imipenem/cilastatin,meropenem, aztreonam, oxytetracycline, azithromycin, clarithromycin,dirithromycin, erythromycin, roxithromycin, spiramycin andciprofloxacin. The alginate oligomers of the invention may therefore beused to increase (or improve) the efficacy (or effectiveness) of theantibiotics described herein, or more particularly the particularizedsubgroups thereof, particularly in inhibiting the growth of bacteria,especially MDR bacteria. For example the dose of the antibiotic beingused together with the alginate oligomers of the invention may belowered as a consequence.

With respect to azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin and spiramycin (or more generally themacrolides as a class of antibiotics) as noted above, data presented inthe Examples show more generally that alginate oligomers may potentiatethe effects of these antibiotics (and this class) against a range ofdifferent bacteria. Alginate oligomers may thus be used to increase (orimprove) the efficacy of these antibiotics (or more generally theantibiotic class of macrolides), for example to enable a lower dose ofthe antibiotic to be used.

Thus, in another aspect the invention provides a method to improve theefficacy of a macrolide antibiotic, and in particular the effectiveness(or efficacy) of a macrolide antibiotic to inhibit the growth and/orviability of bacteria (which includes inhibition of the growth of abacterial population, as well as growth of a bacterium), said methodcomprising using said antibiotic together with (in conjunction orcombination with) an alginate oligomer (which may be any alginateoligomer as defined herein). More particularly, the using step maycomprise contacting the bacteria with an alginate oligomer at the sameor substantially the same time or prior to contacting the bacteria withthe macrolide antibiotic. In particular, and in accordance with thedisclosures made herein (and specifically the definitions providedherein), which can be read as applying to all aspects of the presentinvention, the step of contacting the bacterium with the alginateoligomer may include administering the alginate oligomer to a subject.Conveniently the macrolide antibiotic is applied or administeredsimultaneously with the oligomer or almost immediately before or afterthe oligomer. However the macrolide antibiotic may be applied oradministered at least 1 hour, at least 3 hours, at least 6 hours afterthe oligomer. In these embodiments the macrolide antibiotic can beapplied or administered with or without a further application of analginate oligomer. The oligomer can be applied or administered in aplurality of applications prior to or with the macrolide antibiotic.Other dosing regimes (e.g. where the antibiotic is administered beforethe oligomer) are described in more detail above and apply mutatismutandis to this aspect of the invention.

The macrolide antibiotic may be selected from the group azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin,telithromycin, CarbomycinA, josamycin, kitasamycin, midecamicine,oleandomycin, spiramycin, troleandromycin, tylosin. Preferably themacrolide antibiotic is an azalide macrolide, preferably azithromycin.The bacterium can be from any family, genus or species of bacteria (e.g.it may be any of the bacteria discussed and preferred above). Preferablyit is an MDR bacterium as defined above. Preferably it is selected fromthe group Pseudomonas (e.g. Pseudomonas aeruginosa), Staphylococcus(e.g. Staphylococcus aureus), Streptococcus (e.g. Streptococcusagalactiae, Streptococcus pneumoniae, Streptococcus pyogenes),Haemophilus (e.g. Haemophilus ducreyi, Haemophilus influenzae),Moraxella (e.g. Moraxella catarrhalis), Neisseria (e.g. Neisseriagonorrhoeae), Chlamydia (e.g. Chlamydia pneumoniae, Chlamydiatrachomatis), Mycoplasma (e.g. Mycoplasma pneumoniae), Helicobacter(e.g. Helicobacter pylori), Salmonella (e.g. Salmonella typhi)Burkholderia (e.g. Burkholderia cepacia, Burkholderia mallei,Burkholderia pseudomallei), Acinetobacter (e.g. Acinetobacter baumannii,Acinetobacter lwoffi), Providencia (e.g. Providencia stuartii,Providencia rettgeri, Providencia alcalifaciens), and Klebsiella (e.g.Klebsiella oxytoca).

The location of the bacterium or population is not restricted (e.g. itmay be any of the locations discussed and preferred below). In oneembodiment the bacterium or population will not be in a biofilm. In oneembodiment the bacterium or population will be in a biofilm. Thus, themethod may be an in vitro or an in vivo method. In the latter instancethe method can be viewed as a method for the treatment of a bacterialinfection in a subject (e.g. those bacterial infections and subjectsdescribed and preferred above or elsewhere herein), said methodcomprising administering to a subject a pharmaceutically effectiveamount of an alginate oligomer at substantially the same time as orprior to administering a pharmaceutically effective amount of amacrolide antibiotic.

Thus the invention provides an alginate oligomer for use together with(or in combination or conjunction with) a macrolide antibiotic for thetreatment of a bacterial infection in a subject. “Use together” is asdefined above.

Alternatively put; the invention provides the use of an alginateoligomer for the manufacture of a medicament for use together with amacrolide antibiotic in the treatment of a bacterial infection in asubject. The medicament may further comprise the macrolide antibiotic.

The medicament may be in the form of a single composition or formulationcomprising the alginate oligomer and macrolide antibiotic(s) or separatecompositions or formulations may be prepared and used, each containingthe alginate oligomer or the macrolide antibiotic(s), respectively.

Thus in a more particular aspect the present invention provides the useof an alginate oligomer and at least one macrolide antibiotic for themanufacture of a medicament for use in the treatment of a bacterialinfection in a subject.

Thus a further aspect of the present invention provides a productcontaining an alginate oligomer and a macrolide antibiotic (or one ormore macrolide antibiotics) as a combined preparation for separate,simultaneous or sequential use in the treatment of a bacterial infectionin a subject.

As noted above, in these aspects of the invention the alginate oligomermay improve the efficacy of the antibiotic, and in particular theefficacy (or effectiveness) of the antibiotic in inhibiting bacterialgrowth.

Improving the efficacy of the antibiotic includes any aspect ofimproving or enhancing the effect of the antibiotic, e.g. so that theanti-bacterial effect of the antibiotic is increased or enhanced in anyway over the effect of the antibiotic seen in the absence of thealginate oligomer. This may be seen for example in a stronger effect ofthe antibiotic in inhibiting growth of the bacteria, requiring lessantibiotic to achieve the same effect seen in the absence of alginateoligomer, or a increased effectiveness seen as increased speed or rateof action, an inhibitory effect being seen in less time than in theabsence of oligomer.

The references to “improving the effectiveness of a macrolide antibioticto inhibit the growth and/or viability of bacteria” etc. accordingly mayinclude that the alginate oligomer renders the macrolide antibiotic, atleast twice as, or at least four times, at least eight times, at leastsixteen times or at least thirty two times more effective at inhibitingbacterial growth (e.g. acting as a bacteriostatic agent). Put in adifferent way, the oligomer may at least double, at least quadruple, atleast octuple, at least sexdecuple or at least duotrigenuple theeffectiveness of the macrolide antibiotic to inhibit growth of thebacteria. The inhibitory effect of the macrolide antibiotic can bemeasured by assessing the Minimum Inhibitory Concentration (MIC), i.e.that concentration of macrolide antibiotic that completely inhibitsgrowth of the bacteria. A halving of the MIC corresponds to a doublingin the inhibitory effect of the macrolide antibiotic. A quartering ofthe MIC corresponds to a quadrupling of the inhibitory effect.

This aspect also allows the concentration of the macrolide antibioticadministered to a subject or applied to a location to be reduced whilstmaintaining the same effectiveness. This can be beneficial if themacrolide antibiotic is expensive or associated with side effects.Minimising the use of antibiotics is also desirable to minimisedevelopment of resistance. In accordance with the invention the use ofan alginate oligomer as described above, i.e. at the same orsubstantially the same time or prior to administering the macrolideantibiotic permits the antibiotic to be used at a concentration that isless than 50%, less than 25%, less than 10% or less than 5% of theamount normally administered/applied to achieve a particular level ofinhibition of the growth of bacteria in the absence of the alginateoligomer.

In this aspect the alginate oligomers may be any of those discussed andin particular those stated as preferred above and the alginate oligomerswill be applied to the bacteria and/or their location at a localconcentration of at least 2%, at least 4%, at least 6%, at least 8% orat least 10% weight by volume.

Alginate oligomers may similarly potentiate the effects of ciprofloxacin(and the quinolones as a class of antibiotics) and aztreonam (and theβ-lactams, e.g. the monobactams as a class of antibiotics), and may thusbe used to increase (or improve) the efficacy of these antibiotics (ormore generally the antibiotic classes of quinolones and β-lactams, e.g.the monobactams), for example to enable a lower dose of theseantibiotics to be used. Accordingly, alginate oligomers may be usedanalogously to as described above for macrolide antibiotics to increasethe efficacy of these antibiotics and the statements made above in thecontext of macrolides apply analogously to the quinolone and/or β-lactamantibiotic classes also.

In the context of the “MDR” aspects of the invention, in otherembodiments the MDR bacterium targeted by the method of the invention isa bacterium which is resistant to at least one antibiotic that is aconventional or standard (e.g. clinically approved) treatment for (oragainst) that bacterium. The skilled man would be aware of theconventional and recommended antibiotics for the treatment of anyparticular bacterial infection or disease. Factors that dictate what isa conventional treatment are well known to the skilled man and includethe nature and location of the bacterium, the intrinsic susceptibilityof the bacterium, the necessary route of administration and theconsequent pharmacokinetics of the antibiotics. Typically an antibioticwhich is a conventional treatment for a bacterium will be an antibioticto which a reference (i.e. typical or wild type) bacterium of thatspecies displays no intrinsic resistance in vitro and/or in the clinicalsetting. As discussed below, the skilled man would be able to employroutine assays to determine this information for any antibiotic orbacterium he could not obtain standard information for from theliterature or his common general knowledge.

Alternatively defined, in certain embodiments the MDR bacterium targetedin accordance with the invention is a bacterium which has acquired(developed) some or all of its antibiotic resistance. Particularly suchantibiotic resistance is acquired in a clinical setting. Particularstrains of bacteria which have acquired multiple antibiotic resistanceare sometimes termed MDR strains as their resistant phenotype differsfrom that of a corresponding strain (e.g. the wild-type strain or a“typical” strain) which has not acquired multidrug resistance, butdemonstrates only the innate or intrinsic resistance which is typical ofthe species. Therefore, in particularly preferred embodiments thebacterium targeted by the invention is a bacterium from an MDR strain ofa species of bacteria (e.g. a strain known or identified in the art asMDR). In these embodiments the MDR bacterium (e.g. bacterium from an MDRstrain of bacteria) targeted by the method of the invention has acquiredor developed resistance to at least 1, e.g. at least 2, 3, 4, 5, 6, 7,8, 9 or 10 structurally and/or functionally different antibiotics orantibiotic classes. In some cases, all of the antibiotic resistance ofthe bacterium (e.g. bacterium from an MDR strain of bacteria) isacquired or developed and none of the resistance is intrinsic, but asnoted above, it is not necessarily the case that an MDR phenotype isacquired, and the MDR bacterium which is treated according to thepresent invention may be MDR intrinsically (or innately).

The MDR bacterium targeted according to the invention can be selectedfrom any genera or species of bacteria. Examples of genera or species ofbacteria include, but are not limited to, Abiotrophia, Achromobacter,Acidaminococcus, Acidovorax, Acinetobacter, Actinobacillus,Actinobaculum, Actinomadura, Actinomyces, Aerococcus, Aeromonas, Afipia,Agrobacterium, Alcaligenes, Alloiococcus, Alteromonas, Amycolata,Amycolatopsis, Anaerobospirillum, Anaerorhabdus, Arachnia,Arcanobacterium, Arcobacter, Arthrobacter, Atopobium, Aureobacterium,Bacteroides, Balneatrix, Bartonella, Bergeyella, Bifidobacterium,Bilophila Branhamella, Borrelia, Bordetella, Brachyspira, Brevibacillus,Brevibacterium, Brevundimonas, Brucella, Burkholderia, Buttiauxella,Butyrivibrio, Calymmatobacterium, Campylobacter, Capnocytophaga,Cardiobacterium, Catonella, Cedecea, Cellulomonas, Centipeda, Chlamydia,Chlamydophila, Chromobacterium, Chyseobacterium, Chryseomonas,Citrobacter, Clostridium, Collinsella, Comamonas, Corynebacterium,Coxiella, Cryptobacterium, Delftia, Dermabacter, Dermatophilus,Desulfomonas, Desulfovibrio, Dialister, Dichelobacter, Dolosicoccus,Dolosigranulum, Edwardsiella, Eggerthella, Ehrlichia, Eikenella,Empedobacter, Enterobacter, Enterococcus, Erwinia, Erysipelothrix,Escherichia, Eubacterium, Ewingella, Exiguobacterium, Facklamia,Filifactor, Flavimonas, Flavobacterium, Francisella, Fusobacterium,Gardnerella, Globicatella, Gemella, Gordona, Haemophilus, Hafnia,Helicobacter, Helococcus, Holdemania, lgnavigranum, Johnsonella,Kingella, Klebsiella, Kocuria, Koserella, Kurthia, Kytococcus,Lactobacillus, Lactococcus, Lautropia, Leclercia, Legionella,Leminorella, Leptospira, Leptotrichia, Leuconostoc, Listeria,Listonella, Megasphaera, Methylobacterium, Microbacterium, Micrococcus,Mitsuokella, Mobiluncus, Moellerella, Moraxella, Morganella,Mycobacterium, Mycoplasma, Myroides, Neisseria, Nocardia, Nocardiopsis,Ochrobactrum, Oeskovia, Oligella, Orientia, Paenibacillus, Pantoea,Parachlamydia, Pasteurella, Pediococcus, Peptococcus,Peptostreptococcus, Photobacterium, Photorhabdus, Plesiomonas,Porphyrimonas, Prevotella, Propionibacterium, Proteus, Providencia,Pseudomonas, Pseudonocardia, Pseudoramibacter, Psychrobacter, Rahnella,Ralstonia, Rhodococcus, Rickettsia Rochalimaea Roseomonas, Rothia,Ruminococcus, Salmonella, Selenomonas, Serpulina, Serratia, Shewenella,Shigella, Simkania, Slackia, Sphingobacterium, Sphingomonas, Spirillum,Staphylococcus, Stenotrophomonas, Stomatococcus, Streptobacillus,Streptococcus, Streptomyces, Succinivibrio, Sutterella, Suttonella,Tatumella, Tissierella, Trabulsiella, Treponema, Tropheryma,Tsakamurella, Turicella, Ureaplasma, Vagococcus, Veillonella, Vibrio,Weeksella, Wolinella, Xanthomonas, Xenorhabdus, Yersinia, and Yokenella;e.g. gram-positive bacteria such as, M. tuberculosis, M. bovis, M.typhimurium, M. bovis strain BCG, BCG substrains, M. avium, M.intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans, M.avium subspecies paratuberculosis, Staphylococcus aureus, Staphylococcusepidermidis, Staphylococcus equi, Streptococcus pyogenes, Streptococcusagalactiae, Listeria monocytogenes, Listeria ivanovii, Bacillusanthracis, B. subtilis, Nocardia asteroides, Actinomyces israelii,Propionibacterium acnes, Clostridium tetani, Clostridium perfringens,Clostridium botulinum, and Enterococcus species and Gram-negativebacteria such as Pseudomonas aeruginosa, Vibrio cholerae, Actinobacilluspleuropneumoniae, Pasteurella haemolytica, Pasteurella multocida,Legionella pneumophila, Salmonella typhi, Brucella abortus, Coxiellaburnetti, Escherichia coli, Neiserria meningitidis, Neiserria gonorrhea,Haemophilus influenzae, Haemophilus ducreyi, Yersinia pestis, Yersiniaenterolitica, Escherichia hirae, Burkholderia cepacia, Burkholderiamallei, Burkholderia pseudomallei, Francisella tularensis, Bacteroidesfragilis, Fusobascterium nucleatum, Cowdria ruminantium, Moraxellacatarrhalis, Klebsiella pneumoniae, Proteus mirabilis, Enterobactercloacae, Serratia marcescens, Helicobacter pylori, Salmonellaenteritidis, Salmonella typhi and Acinetobacter baumannii, Acinetobacterlwoffi, Providencia stuartii, Providencia rettgeri, Providenciaalcalifaciens and Klebsiella oxytoca and Gram non-responsive bacteriasuch as Chlamydia trachomatis, Chlamydia psittaci.

Preferably the MDR bacterium targeted according to the invention isselected from the following genera: Achromobacter, Acinetobacter,Actinobacillus, Aeromonas, Agrobacterium, Alcaligenes, Alteromonas,Bacteroides, Bartonella, Borrelia, Bordetella, Brucella, Burkholderia,Campylobacter, Cardiobacterium, Chlamydia, Chlamydophila,Chromobacterium, Chyseobacterium, Chryseomonas, Citrobacter,Clostridium, Comamonas, Corynebacterium, Coxiella, Cryptobacterium,Edwardsiella, Eikenella, Enterobacter, Enterococcus, Erwinia, Kingella,Klebsiella, Lactobacillus, Lactococcus, Legionella, Leptospira,Leptotrichia, Leuconostoc, Listeria, Listonella, Mobiluncus, Moraxella,Morganella, Mycobacterium, Mycoplasma, Neisseria, Nocardia,Nocardiopsis, Pantoea, Parachlamydia, Pasteurella, Peptococcus,Peptostreptococcus, Prevotella, Propionibacterium, Proteus, Providencia,Pseudomonas, Ralstonia, Rickettsia, Salmonella, Shewenella, Shigella,Sphingobacterium, Sphingomonas, Staphylococcus, Stenotrophomonas,Streptobacillus, Streptococcus, Streptomyces, Treponem and Yersinia

As noted above, the invention includes both medical and non-medical usesand hence the bacteria which may be treated or combatted according tothe present invention include not only clinically-relevant strains, butany bacteria which may present a problem of colonisation orcontamination. In certain aspects clinically-relevant genera, species orstrains of bacteria are preferred.

In certain embodiments the MDR bacterium is selected from the genera,Acinetobacter, Klebsiella, Providencia, Pseudomonas and Burkholderia,e.g. the bacterium is from a species selected from Acinetobacterbaumannii, Acinetobacter baylyi, Acinetobacter bouvetii, Acinetobactercalcoaceticus, Acinetobacter gerneri, Acinetobacter grimontii,Acinetobacter haemolyticus, Acinetobacter johnsonii, Acinetobacterjunii, Acinetobacter lwoffii, Acinetobacter parvus, Acinetobacterradioresistens, Acinetobacter schindleri, Acinetobacter tandoii,Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacterursingii, Klebsiella granulomatis, Klebsiella oxytoca, Klebsiellapneumoniae, Klebsiella singaporensis, Klebsiella variicola, Providenciastuartii, Providencia sneebia, Providencia rettgeri, Providenciarustigianii, Providencia heimbachae, Providencia burhodogranariea,Providencia alcalifaciens, Pseudomonas aeruginosa, Pseudomonasalcaligenes, Pseudomonas anguilliseptica, Pseudomonas argentinensis,Pseudomonas borbori, Pseudomonas citronellolis, Pseudomonas flavescens,Pseudomonas mendocina, Pseudomonas nitroreducens, Pseudomonasoleovorans, Pseudomonas pseudoalcaligenes, Pseudomonas resinovorans,Pseudomonas straminea, Pseudomonas cremoricolorata, Pseudomonas fulva,Pseudomonas monteilii, Pseudomonas mosselii, Pseudomonas oryzihabitans,Pseudomonas parafulva, Pseudomonas plecoglossicida, Pseudomonas putida,Pseudomonas balearica, Pseudomonas luteola, and Pseudomonas stutzeri,Burkholderia ambifaria, Burkholderia andropogonis, Burkholderia anthina,Burkholderia brasilensis, Burkholderia calcdonica, Burkholderiacaribensis, Burkholderia caryophylli, Burkholderia cenocepacia,Burkholderia cepacia, Burkholderia dolosa, Burkholderia fungorum,Burkholderia gladioli, Burkholderia glathei, Burkholderia glumae,Burkholderia graminis, Burkholderia hospita, Burkholderia kururiensis,Burkholderia mallei, Burkholderia multivorans, Burkholderia phenazinium,Burkholderia phenoliruptrix, Burkholderia phymatum, Burkholderiaphytofirmans, Burkholderia plantarii, Burkholderia pseudomallei,Burkholderia pyrrocinia, Burkholderia sacchari, Burkholderiasingaporensis, Burkholderia sordidicola, Burkholderia stabilis,Burkholderia terricola, Burkholderia thailandensis, Burkholderiatropica, Burkholderia tuberum, Burkholderia ubonensis, Burkholderiaunamae, Burkholderia vietnamiensis, and Burkholderia xenovorans. TheBurkholderia species are of particular note, especially Burkholderiacepacia, Burkholderia pseudomallei and Burkholderia mallei; e.g.Burkholderia cepacia.

Thus, the invention may be used against Gram positive or Gram negativebacteria, or indeed Gram-indeterminate bacteria. Gram-negative bacteria,for instance those particularized above, are of importance. Within theGram-negative bacteria the Enterobacteriaceae and the Gram-negativebacteria non-fermenting bacteria are of particular note.

Enterobacteriaceae include, but are not limited to, bacteria from thegenera Alishewanella, Alterococcus, Aquamonas, Aranicola, Azotivirga,Brenneria, Budvicia, Buttiauxefia, Cedecea, Citrobacter, Cronobacter,Dickeya, Edwardsiella, Enterobacter, Erwinia, Escherichia, Ewingella,Grimontella, Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorella,Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium,Phlomobacter, Photorhabdus, Plesiomonas, Pragia, Proteus, Providencia,Rahnella, Raoultella, Salmonella, Samsonia, Serratia, Shigella, Sodalis,Tatumella, Trabulsiella, Wigglesworthia, Xenorhabdus, Yersinia,Yokenella. Preferred genera of Enterobacteriaceae include Escherichia,Klebsiella, Salmonella, Shigella, Yersinia and Providencia.

Non-fermenting Gram-negative bacteria include, but are not limited to,bacteria from the genera Pseudomonas, Acinetobacter, Stenotrophomonasand Burkholderia, Achromobacter, Algaligenes, Bordetella, Brevundimonas,Comamonas, Elizabethkingia (formerly Chryseobacterium),Methylobacterium, Moraxella, Ochrobactrum, Oligella, Psychrobacter,Ralstonia, Roseomonas, Shewanella, Sphingobacterium, e.g. Pseudomonasaeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, andBurkholderia spp.

Preferably the bacteria may be selected from the genera Pseudomonas,Acinetobacter, Stenotrophomonas, Burkholderia, Escherichia, Klebsiella,Providencia, Streptococcus, Staphylococcus, e.g. Pseudomonas aeruginosa,Acinetobacter baumannii, Stenotrophomonas maltophilia, Burkholderia spp,E. coli, Klebsiella pneumoniae and Burkholderia cepacia, Burkholderiamallei, Burkholderia pseudomallei, Acinetobacter lwoffi, Providenciastuartii, Providencia rettgeri, Providencia alcalifaciens, Klebsiellaoxytoca, Pseudomonas anguilliseptica, Pseudomonas oryzihabitans,Pseudomonas plecoglossicida, Pseudomonas luteola, and MRSA.

Results presented in the Examples below show particularly that alginateoligomers may be used together with various antibiotics against MDRstrains of Pseudomonas and particularly MDR strains of P. aeruginosa.The results also show that alginate oligomers may effectively be usedwith various antibiotics against Acinetobacter species, and particularlyA. baumannii and A. lwoffii; against Burkholderia species, andparticularly B. cepacia; against Providencia species, and particularlyP. stuartii; against Klebsiella species, and particularly Klebsiellapneumonia; against Streptococcus, and particularly Streptococcus oralis;against Staphylococcus, and in particular MRSA; against Escherichia, andparticularly Escherichia coli, and that resistance to antibiotics inthese genera/species may be overcome.

In this regard, the data more generally show that alginate oligomers maybe particularly effective in potentiating (or improving/increasing theefficacy of) the effects of antibiotics against Acinetobacter species,and particularly A. baumannii and Burkholderia species, and particularlyB. cepacia. This leads to the proposal that in one aspect the inventioncan be seen more generally to relate to use of alginate oligomers inconjunction (or combination) with an antibiotic to combat (or to inhibitthe growth and/or viability of) Acinetobacter and/or Burkholderia (i.e.Acinetobacter and/or Burkholderia species in general), for example totreat or combat infection and/or contamination (i.e. colonisation) withthese bacteria.

In certain aspects, the bacterium targeted by the invention mayalternatively be viewed as a clinically relevant bacterium, e.g. abacterium that is known to be associated with disease and/or infectionin subjects; especially diseases and infections that are unresponsive toat least 3 structurally and/or functionally different antibiotics, or atleast 3 antibiotic classes, more particularly at least 4, 5, 6, 7 8, 9or 10 structurally and/or functionally different antibiotics orantibiotic classes conventionally used in the treatment of that diseaseand/or infection. More particularly, the bacterium targeted by theinvention may be from a clinically relevant MDR strain of bacteria. Thebacterium may cause or result in clinically significant or clinicallyimportant infections, in other words infections which are the cause ofsignificant clinical problems. For instance, the bacterium could be abacterium associated with nosocomial infections, infections in therespiratory tract of patients, e.g. patients suffering from cysticfibrosis, chronic obstructive pulmonary disease, congestive obstructiveairway disease/congestive obstructive airway pneumonia (COAD/COAP),pneumonia, emphysema, bronchitis and sinusitis; infections in chronicwounds (including burns), device related infections associated withimplantable or prosthetic medical devices e.g. prosthetic valveendocarditis or infection of lines or catheters or artificial joints ortissue replacements or endotracheal or tracheotomy tubes. Examples ofthese types of bacteria include Pseudomonas aeruginosa, Acinetobacterbaumannii, Stenotrophomonas maltophilia, Burkholderia spp (e.g. B.cepacia), E. coli, Klebsiella pneumoniae, Staphylococcus aureus,Methicillin Resistant Staphylococcus aureus (MRSA), Clostridiumdifficile, Mycobacterium tuberculosis, Enterococcus andVancomycin-Resistant Enterococcus and Providencia stuartii.

The bacterium targeted by the method of the invention may be the same asa bacterium that has previously been isolated from a subject. Thus, thebacterium is preferably a clinical strain or a clinical isolate. Thebacterium targeted by the method of the invention may be present in oron a subject. The bacterium may be known or found to be MDR, or thebacterium may have developed MDR during the subject's treatment. In viewof the requirement for MDR (or MDR status), which may or may not be orinclude acquired resistance, the bacterium to be treated according tothe present invention will generally not be a conventional laboratory orreference strain, e.g. a strain such as Pseudomonas aeruginosa PA01(ATCC 15692) or Staphylococcus aureus ATCC 6538. In another embodimentthe bacterium will not be MRSA (methicillin resistant Staphylococcusaureus), e.g. strain 1103.

In representative embodiments the bacterium may be an MDR strain ofPseudomonas aeruginosa that is resistant to one or more antibioticsselected from the penicillins, cephalosporins, carbapenems, monobactams,aminoglycosides, fluoroquinolones, macrolides or polypeptides (e.g.polymyxins), more particularly cephalosporins, carbapenems, monobactams,aminoglycosides, fluoroquinolones, or macrolides, e.g. amikacin,ciprofloxacin, gentamicin, tobramycin, piperacillin, ticarcillin,colistin, oxytetracycline, meropenem, ceftazidime, aztreonam,azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, spiramycin and imipenem/cilastatin; particularlyciprofloxacin, colistin, oxytetracycline, meropenem, ceftazidime,aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, spiramycin and imipenem/cilastatin, and especiallyciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycinand imipenem/cilastatin, e.g. aztreonam, ciprofloxacin, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin andspiramycin.

In other embodiments the bacterium may be an MDR strain of Klebsiellapneumoniae that is resistant to one or more antibiotics selected fromthe penicillins, cephalosporins, carbapenems, monobactams,aminoglycosides, fluoroquinolones, macrolides or polypeptides (e.g.polymyxins) e.g. cefotaxime, ceftriaxone, amikacin, gentamicin,ciprofloxacin, tobramycin, ampicillin, piperacillin, ticarcillin,colistin, meropenem, ceftazidime, aztreonam, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin,imipenem/cilastatin; cefepime, levofloxacin, norfloxacin, gatifloxacin,moxifloxacin, and ertapenem, particularly ciprofloxacin, colistin,meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin,dirithromycin, erythromycin, roxithromycin, spiramycin andimipenem/cilastatin, and especially ciprofloxacin, meropenem,ceftazidime, aztreonam, azithromycin and imipenem/cilastatin, e.g.aztreonam, ciprofloxacin, azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin and spiramycin.

In other embodiments the bacterium may be an MDR strain of Acinetobacterbaumannii that is resistant to one or more antibiotics selected from thepenicillins, cephalosporins, carbapenems, monobactams, glycylcyclines,aminoglycosides, fluoroquinolones, macrolides or polypeptides (e.g.polymyxins). e.g. imipenem/cilastatin, ampicillin, cefepime, colistin,rifampin, tigecycline, amikacin, ciprofloxacin, meropenem, ceftazidime,aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, spiramycin and imipenem/cilastatin; particularlycolistin, ciprofloxacin, meropenem, ceftazidime, aztreonam,azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, spiramycin and imipenem/cilastatin, and especiallyciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycinand imipenem/cilastatin, e.g. aztreonam, ciprofloxacin, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin andspiramycin.

In other embodiments the bacterium may be an MDR strain of Providenciastuartii that is resistant to one or more antibiotics selected from thepenicillins, cephalosporins, carbapenems, monobactams, aminoglycosides,fluoroquinolones, macrolides or polypeptides (e.g. polymyxins) e.g.cefotaxime, ceftriaxone, amikacin, gentamicin, ciprofloxacin,tobramycin, ampicillin, piperacillin, ticarcillin, colistin, meropenem,ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, spiramycin, imipenem/cilastatin; cefepime,levofloxacin, norfloxacin, gatifloxacin, moxifloxacin, and ertapenem,particularly ciprofloxacin, colistin, ciprofloxacin, meropenem,ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, spiramycin and imipenem/cilastatin, andespecially ciprofloxacin, meropenem, ceftazidime, aztreonam,azithromycin and imipenem/cilastatin, e.g. aztreonam, ciprofloxacin,azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycinand spiramycin.

In other embodiments the bacterium may be an MDR strain of Burkholderiacepacia that is resistant to one or more antibiotics selected from thepenicillins, cephalosporins, carbapenems, monobactams, aminoglycosides,fluoroquinolones, macrolides or polypeptides (e.g. polymyxins) e.g.cefotaxime, ceftriaxone, amikacin, gentamicin, ciprofloxacin,tobramycin, ampicillin, piperacillin, ticarcillin, colistin, meropenem,ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, spiramycin, imipenem/cilastatin; cefepime,levofloxacin, norfloxacin, gatifloxacin, moxifloxacin, and ertapenem,particularly ciprofloxacin, colistin, ciprofloxacin, meropenem,ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, spiramycin and imipenem/cilastatin, andespecially ciprofloxacin, meropenem, ceftazidime, aztreonam,azithromycin and imipenem/cilastatin, e.g. aztreonam, ciprofloxacin,azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycinand spiramycin.

The data of the Examples surprisingly shows that the alginate oligomersof the invention are particularly effective in enhancing the effects(increasing the effectiveness (or efficacy)) of antibiotics againstbacteria of the genus Burkholderia. As discussed above, Burkholderiarepresent an important genus of bacteria since they can cause disease inhumans and animals and they display intrinsic resistance to multipleclasses of antibiotics (e.g. the aminoglycosides, the β lactams and/orthe macrolides). Burkholderia organisms, especially Burkholderiacepacia, Burkholderia pseudomallei and Burkholderia mallei are thereforeconsidered to be MDR bacteria naturally on account of the intrinsicresistance exhibited as their natural phenotype. Of course, strains ofBurkholderia species can acquire additional resistance phenotypes.Accordingly, treatments for Burkholderia species that enhance theeffects of antibiotics against such species are in high demand.

Thus, it is a preferred embodiment of the invention that the targetbacterium is a Burkholderia organism, e.g. selected from Burkholderiaambifaria, Burkholderia andropogonis, Burkholderia anthina, Burkholderiabrasilensis, Burkholderia calcdonica, Burkholderia caribensis,Burkholderia caryophylli, Burkholderia cenocepacia, Burkholderiacepacia, Burkholderia dolosa, Burkholderia fungorum, Burkholderiagladioli, Burkholderia glathei, Burkholderia glumae, Burkholderiagraminis, Burkholderia hospita, Burkholderia kururiensis, Burkholderiamallei, Burkholderia multivorans, Burkholderia phenazinium, Burkholderiaphenoliruptrix, Burkholderia phymatum, Burkholderia phytofirmans,Burkholderia plantarii, Burkholderia pseudomallei, Burkholderiapyrrocinia, Burkholderia sacchari, Burkholderia singaporensis,Burkholderia sordidicola, Burkholderia stabilis, Burkholderia terricola,Burkholderia thailandensis, Burkholderia tropica, Burkholderia tuberum,Burkholderia ubonensis, Burkholderia unamae, Burkholderia vietnamiensis,and Burkholderia xenovorans, in particular Burkholderia cepacia,Burkholderia mallei and Burkholderia pseudomallei and especiallyBurkholderia cepacia.

More generally, the use of alginate oligomers to combat Burkholderia(e.g. to treat or combat Burkholderia infection and/or contamination(i.e. colonisation)), or to increase (or improve) the efficacy of anantibiotic against Burkholderia represents a particular preferred andseparate aspect of this invention.

Accordingly, in another aspect the invention provides a method toimprove the efficacy of an antibiotic, and in particular theeffectiveness (or efficacy) of an antibiotic to inhibit the growthand/or viability of a Burkholderia organism (which includes inhibitionof the growth of a Burkholderia population, as well as growth of aBurkholderia organism), said method comprising using said antibiotictogether with (in conjunction or combination with) an alginate oligomer(which may be any alginate oligomer as defined herein and especiallythose indicated already as being preferred; e.g. the “high G”, “high M”,“G-block” and “M-block” oligomers). The oligomers of use in this aspectmay especially be those with the sizes, size ranges and molecular weightdistributions stated as preferred above. The discussion of preferredalginate oligomers of the invention applies mutatis mutandis to thisaspect of the invention.

More particularly, the using step may comprise contacting theBurkholderia organism with an alginate oligomer at the same orsubstantially the same time or prior to contacting the Burkholderiaorganism with the antibiotic. In particular, and in accordance with thedisclosures made above (and specifically the definitions providedherein), which can be read as applying to all aspects of the presentinvention, the step of contacting the bacterium with the alginateoligomer may include administering the alginate oligomer and theantibiotic to a subject.

The antibiotic may be selected from any antibiotic disclosed above.Preferred antibiotics may be selected from the β-lactams (e.g. thecarbecephems (e.g. loracarbef); the 1st generation cephalosporins (e.g.cefadroxil, cefazolin, cephalexin); 2nd generation cephalosporins (e.g.cefaclor, cefamandole, cephalexin, cefoxitin, cefprozil, cefuroxime);3rd generation cephalosporins (e.g. cefixime, cefdinir, cefditoren,cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten,ceftizoxime, ceftriaxone); 4th generation cephalosporins (e.g.cefepime); the monobactams (e.g. aztreonam); the penicillins (e.g.amoxicillin, ampicillin, carbenicillin, cloxacillin, dicloxacillin,nafcillin, oxacillin, penicillin G, penicillin V, piperacillin,ticarcillin) the carbapenems (e.g. imipenem, meropenem, ertapenem,doripenem, panipenem/betamipron, biapenem, PZ-601)); the macrolides(e.g. azithromycin, clarithromycin, dirithromycin, erythromycin,troleandomycin); the quinolones (e.g. ciprofloxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,ofloxacin, trovafloxacin); and the tetracyclines (e.g. demeclocycline,doxycycline, minocycline, oxytetracycline, tetracycline).

More preferably the antibiotic is selected from azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin,telithromycin, CarbomycinA, josamycin, kitasamycin, midecamicine,oleandomycin, spiramycin, troleandromycin, tylosin, cefixime, cefdinir,cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime,ceftibuten, ceftizoxime, ceftriaxone, cefepime, aztreonam, imipenem,meropenem, ertapenem, doripenem, panipenem/betamipron, biapenem, PZ-601,ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin,moxifloxacin, norfloxacin, ofloxacin, trovafloxacin demeclocycline,doxycycline, minocycline, oxytetracycline and tetracycline, e.g.aztreonam, ciprofloxacin, azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, spiramycin, oxytetracycline, ceftazidimeand imipenem. In particularly preferred embodiments the antibiotic isselected from aztreonam, ceftazidime, azithromycin, clarithromycin anderythromycin

In other embodiments the antibiotic used is not tobramycin, amikacinand/or colistin. In other embodiments the antibiotic used is not anaminoglycoside or a polypeptide antibiotic. In other embodiments theantibiotic used is not an antibiotic that has a positive charge underthe conditions in which it will be used with the alginate oligomer, e.g.antibiotics with at least 3, e.g. at least 4, 5, 6 or 7 amino (—NH₂)groups.

The Burkholderia organism can be from any Burkholderia species, e.g. anyof those disclosed herein, in particular the Burkholderia organism willbe Burkholderia cepacia, Burkholderia mallei or Burkholderiapseudomallei, especially Burkholderia cepacia. In certain embodimentsthe Burkholderia organism is resistant to the antibiotic.

In one embodiment the Burkholderia organism or population thereof willnot be in a biofilm or in the process of forming a biofilm. In anotherembodiment the Burkholderia organism or population thereof will be in abiofilm.

The method may be an in vitro or an in vivo method and may havenon-medical and medical applications. In the latter instance the methodcan be viewed as a method for the treatment of a Burkholderia infection(e.g. a Burkholderia cepacia, Burkholderia mallei or Burkholderiapseudomallei infection) in a subject (e.g. those subjects described andpreferred herein), said method comprising administering to a subject apharmaceutically effective amount of an alginate oligomer together witha pharmaceutically effective amount of an antibiotic. This embodimentextends to any and all of the medical uses, diseases and locations ofinfection described herein when involving Burkholderia organisms.

Thus the invention provides an alginate oligomer for use together with(or in combination or conjunction with) an antibiotic for the treatmentor prevention of a Burkholderia infection in a subject. The subject maybe infected, suspected to be infected, or at risk of infection withBurkholderia. The Burkholderia may be of any species mentioned herein.The term “use together” should be interpreted as discussed above.

Alternatively put, the invention provides the use of an alginateoligomer for the manufacture of a medicament for use together with anantibiotic in the treatment of a Burkholderia infection in a subject.The medicament may further comprise the antibiotic.

The medicament may be in the form of a single composition or formulationcomprising the alginate oligomer and antibiotic(s) or separatecompositions or formulations may be prepared and used, each containingthe alginate oligomer or the antibiotic(s), respectively.

Thus in a more particular aspect the present invention provides the useof an alginate oligomer and at least one antibiotic for the manufactureof a medicament for use in the treatment of a Burkholderia infection ina subject.

Thus a further aspect of the present invention provides a productcontaining an alginate oligomer and an antibiotic (or antibiotics) as acombined preparation for separate, simultaneous or sequential use in thetreatment of a Burkholderia infection in a subject.

In a further embodiment of this aspect of the invention there isprovided a method for combating contamination of a site with aBurkholderia organism, said method comprising contacting the site and/orthe Burkholderia organism with (an effective amount of) an alginateoligomer together with (an effective amount of) at least one antibiotic.Such a method may particularly be an in vitro method, and the site maybe any surface or location discussed herein.

As noted above, in these aspects of the invention the alginate oligomermay improve the efficacy of the antibiotic, and in particular theefficacy (or effectiveness) of the antibiotic in inhibiting the growthof the Burkholderia organism.

The references to “improving the effectiveness of an antibiotic toinhibit the growth and/or viability of a Burkholderia organism” shouldbe construed in accordance with preceding discussion of what is meant by“improving the effectiveness of a macrolide antibiotic to inhibit thegrowth and/or viability of bacteria”.

This aspect also allows the concentration of the antibiotic administeredto a subject or applied to a location in order to combat a Burkholderiaorganism to be reduced whilst maintaining the same effectiveness. Thiscan be beneficial if the antibiotic is expensive or associated with sideeffects. Minimising the use of antibiotics is also desirable to minimisedevelopment of resistance. In accordance with the invention the use ofan alginate oligomer together with an antibiotic permits the antibioticto be used at a concentration that is less than 50%, less than 25%, lessthan 10% or less than 5% of the amount normally administered/applied toachieve a particular level of inhibition of the growth of a Burkholderiaorganism in the absence of the alginate oligomer.

In this aspect the alginate oligomers may be any of those discussed andin particular those stated as preferred above and the alginate oligomerswill be applied to the Burkholderia organism and/or their location at alocal concentration of at least 2%, at least 4%, at least 6%, at least8% or at least 10% weight by volume.

Although in certain aspects of the invention as discussed above, thebacterium may be Acinetobacter, in certain particular embodiments theMDR bacterium targeted by the methods of the invention is notAcinetobacter baumannii, or any Acinetobacter species. In anotherembodiment the antibiotic used against the Acinetobacter baumannii, orany Acinetobacter species, is not azithromycin, or any macrolide.

By “resistant to an antibiotic” it is meant that the bacterium displaysa substantially greater tolerance (reduced susceptibility) to anantibiotic as compared to a reference bacterium sensitive to theantibiotic or a typical, or a wild type, version of the bacterium. Sucha substantially greater tolerance may be a statistically significantdecrease in susceptibility to the antibiotic, as measured for example instandard assays, such as MIC assays. In some cases, a bacterium can becompletely unaffected by exposure to an antibiotic. In this instance thebacterium can be considered fully resistant to that antibiotic.

A suitable reference bacterium is Oxford Staphylococcus aureus (NCTC6571) although many others are known in the art and are readilyavailable. Typical, or wild type, versions of a bacterium can beobtained easily from laboratories and culture collections throughout theworld.

Susceptibility (and conversely resistance and tolerance) to antibioticcan be measured in any convenient way, e.g. with dilution susceptibilitytests and/or disk diffusion tests. The skilled man would appreciate thatthe extent of the difference in tolerance/susceptibility sufficient toconstitute resistance will vary depending on the antibiotic and organismunder test and the test used However, a resistant bacterium willpreferably be at least twice, e.g. at least 3, 4, 5, 6, 10, 20, or 50times as tolerant to the antibiotic as the reference bacterium sensitiveto the antibiotic or a typical or a wild type version of the bacterium.Preferably resistance of a particular bacteria to an antibiotic isdetermined using bacteria which are not in a biofilm or which do nothave a biofilm phenotype.

The minimum inhibitory concentration (MIC) assay (Jorgensen et al.,Manual of Clinical Microbiology, 7th ed. Washington, D.C.: AmericanSociety for Microbiology, 1999; 1526-43) is a convenient dilutionsusceptibility test to use. This assay measures the relevant toleranceof a bacterium to antibiotics by determining the lowest concentration ofantibiotic that causes complete inhibition of growth. A bacteriumresistant to an antibiotic will have a substantially greater MIC valuefor the antibiotic than that of the reference bacterium sensitive to theantibiotic or a typical, or a wild type, version of the bacterium, e.g.the resistant bacteria will have a MIC value for the antibiotic that isat least twice or at least four times, at least eight times, at leastsixteen times, at least thirty two times or at least sixty four timeshigher. Put in a different way, the MIC value of the resistant bacteriumfor the antibiotic may be at least double, at least quadruple, at leastoctuple, at least sexdecuple or at least duotrigenuple the MIC value ofthe reference bacterium sensitive to the antibiotic or a typical or awild type version of the bacterium.

Viewed alternatively, and in the context of an in vivo bacterium and/orthe treatment of a bacterial infection that is resistant to multipleantibiotic classes, a bacterium may be considered resistant to anantibiotic if the bacterium has a MIC value for the antibiotic that isgreater than then maximum safe circulating concentration of theantibiotic in the subject (which may be determined easily by the skilledman). More functionally, a bacterium is resistant to an antibiotic if aninfection associated with that bacterium is unresponsive (i.e. there isno change in the clinical indicia of the infection) to the maximum safedose of the antibiotic.

“Overcoming resistance” should be construed accordingly as a measurablereduction in the above-described indicators of the resistance (ormeasurable increase in susceptibility or measurable decrease intolerance) to the antibiotic displayed by the bacterium. Therefore“overcoming resistance” can alternatively be expressed as “reducingresistance”. It is a reference to the observed phenotype of the targetbacterium and should not necessarily be considered to equate to areversal, to any extent, at the mechanistic level of any particularresistance mechanism. As can be seen from the Examples, alginateoligomers and antibiotics have a combinatorial, e.g. synergistic, effectthat makes bacteria with a phenotype that is resistant to an antibioticmore susceptible to that antibiotic. In one embodiment the alginateoligomer will measurably reduce the MIC value of the resistant bacteriumto the antibiotic, e.g. the MIC value will be at least 50%, 25%, 20%,15%, 10%, 5%, 2% or 1% of the MIC value of the bacteria for theantibiotic before treatment in accordance with the invention.

Thus use of alginate oligomers according to the present invention maypotentiate the effect of an antibiotic (or increase or improve itsefficacy). It may render usable (or effective) an antibiotic previouslythought not to be usable/effective against a particular organism, or anantibiotic which is not normally effective against a given organism(e.g. bacterium or bacterial species in question). It may also enable anantibiotic to be used at a reduced dose.

The effects of alginate oligomers in overcoming resistance toantibiotics or in potentiating (etc.) the effects of antibiotics may beseen irrespective of the mechanism of resistance to the antibiotic inquestion. Nevertheless, particularly good results have been observedwith ciprofloxacin. Resistance to this antibiotic may involveaccumulation of mutations, in particular in the genes encoding DNAgyrase or topoisomerase IV. Without wishing to be bound by theory, thealginate oligomers of the invention may therefore affect thisaccumulation process, e.g. by preventing, slowing or halting it.However, it is not to assumed from or implied by this, that alginateoligomers may have any effect on any mechanism of resistance.

In a preferred embodiment of the method of the invention the alginateoligomer overcomes resistance to at least two, e.g. at least 3, 4, 5, 6,7, 8, 9, 10 or all of the structurally and/or functionally differentantibiotics or antibiotic classes to which the bacterium is resistant.However, as noted above, it is not required, or implied, that all of theresistance of any given MDR strain is overcome. The invention may forexample be effective in overcoming resistance to certain classes ofantibiotic in a given MDR strain (e.g. to macrolides and/or quinolonesand/or β-lactams) and this may be clinically useful, even thoughresistance to other antibiotics may remain. This embodiment willpreferably entail the use of a plurality of antibiotics corresponding innumber and identity to some or all of the antibiotic resistancesovercome.

In other embodiments the method of the invention overcomes resistance inan MDR bacterium (e.g. a bacterium from an MDR strain of bacteria) to atleast one antibiotic that is a conventional treatment of that bacterium.Put differently, the method of the invention may overcome resistance inan MDR bacterium (e.g. bacterium from an MDR strain of bacteria) to anantibiotic to which that bacterium has acquired or developed resistance.In these embodiments the method of the invention overcomes at least oneacquired resistance in an MDR bacterium (e.g. bacterium from an MDRstrain of bacteria) that has acquired resistance to at least one, e.g.at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 structurally and/or functionallydifferent antibiotics or antibiotic classes. Preferably all of theacquired antibiotic resistance of the bacterium is overcome. It will beclear to the skilled reader that the invention therefore makes possiblethe treatment of an MDR bacterium (e.g. bacterium from an MDR strain ofbacteria) with an antibiotic that had become ineffective in thetreatment of that bacterium. However, as noted above, not all resistancein an MDR phenotype may be acquired and the invention is not limited tothis. Thus the invention may be used in the treatment of bacteria thatare innately MDR.

The method of the invention may entail contacting the bacterium withmore than one antibiotic. The additional antibiotic(s) can be anyantibiotic, e.g. those listed above. The additional antibiotic(s) may bean antibiotic to which the bacterium is susceptible. The additionalantibiotic(s) may be an antibiotic to which the bacterium is resistant.The additional antibiotic(s) may be used together with (in conjunctionor combination with) the first or other antibiotics and/or the alginateoligomer. More particularly, the step of using may comprise contactingthe bacterium with an alginate oligomer at the same or substantially thesame time or prior to contacting the bacterium with some or all of theantibiotics in an amount effective to overcome the resistance of thebacteria to the antibiotic(s).

As noted above the antibiotic(s) may conveniently be applied oradministered simultaneously with the alginate oligomer, or immediatelyor almost immediately before or after the alginate oligomer. However theantibiotic(s) may be applied or administered at a different time pointe.g. least 1 hour, at least 3 hours, at least 6 hours after the alginateoligomer. It is within the skill of the medical practitioner to developdosage regimes which optimise the effect of the alginate oligomer andantibiotic. In these embodiments the antibiotic(s) can be applied oradministered with or without a further application of an alginateoligomer. The alginate oligomer can be applied or administered in aplurality of applications prior to or with the antibiotic(s). In otherembodiments the antibiotic(s) may conveniently be applied oradministered before the alginate oligomer, e.g. at least 1 hour, atleast 3 hours, at least 6 hours before the alginate oligomer. In theseembodiments the alginate oligomer can be applied or administered with orwithout a further application of the antibiotic(s). The antibiotic(s)can be applied or administered in a plurality of applications prior toor with the alginate oligomer. The skilled man can easily determine whatwould be an appropriate dosing regime for the alginate oligomer andantibiotic(s) he intends to use.

Preferred antibiotic combinations can be two or more from colistin,ciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycinand imipenem/cilastatin, amikacin, gentamicin, oxytetracycline,tobramycin and vancomycin. More particularly, these may be selected fromciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycinimipenem/cilastatin or oxytetracycline, and still more particularly fromciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycinand imipenem/cilastatin.

In preferred embodiments the bacteria is an MDR Acinetobacter,Klebsiella, or Pseudomonas (e.g. Acinetobacter baumannii, Klebsiellapneumoniae, or Pseudomonas aeruginosa) resistant to ceftazidime,ciprofloxacin and azithromycin and the antibiotics used are ceftazidimeor ciprofloxacin together with azithromycin or all of ceftazidime,ciprofloxacin and azithromycin.

The location of the bacterium which may targeted in any aspect of thepresent invention is not restricted, and thus as indicated above, notonly are medical uses covered, but also non-medical uses where thebacterium is not present on or within a clinical subject, but may forexample be present at an abiotic location i.e. the invention may becarried out in vitro. The bacterium may be present on a surface. Thesurface is not limited and includes any surface on which a bacterium mayoccur. The surface may be biotic or abiotic, and inanimate (or abiotic)surfaces include any such surface which may be exposed to microbialcontact or contamination. Thus particularly included are surfaces onmedical equipment, or machinery, e.g. industrial machinery, or anysurface exposed to an aquatic environment (e.g. marine equipment, orships or boats or their parts or components), or any surface exposed toany part of the environment, e.g. pipes or on buildings. Such inanimatesurfaces exposed to microbial contact or contamination include inparticular any part of: food or drink processing, preparation, storageor dispensing machinery or equipment, air conditioning apparatus,industrial machinery, e.g. in chemical or biotechnological processingplants, storage tanks, medical or surgical equipment and cell and tissueculture equipment. Any apparatus or equipment for carrying ortransporting or delivering materials is susceptible to microbialcontamination. Such surfaces will include particularly pipes (which termis used broadly herein to include any conduit or line). Representativeinanimate or abiotic surfaces include, but are not limited to foodprocessing, storage, dispensing or preparation equipment or surfaces,tanks, conveyors, floors, drains, coolers, freezers, equipment surfaces,walls, valves, belts, pipes, air conditioning conduits, coolingapparatus, food or drink dispensing lines, heat exchangers, boat hullsor any part of a boat's structure that is exposed to water, dentalwaterlines, oil drilling conduits, contact lenses and storage cases.

As noted above, medical or surgical equipment or devices represent aparticular class of surface on which bacterial contamination may form.This may include any kind of line, including catheters (e.g. centralvenous and urinary catheters), prosthetic devices, e.g., heart valves,artificial joints, false teeth, dental crowns, dental caps and softtissue implants (e.g. breast, buttock and lip implants). Any kind ofimplantable (or “in-dwelling”) medical device is included (e.g. stents,intrauterine devices, pacemakers, intubation tubes (e.g. endotracheal ortracheostomy tubes), prostheses or prosthetic devices, lines orcatheters). An “in-dwelling” medical device may include a device inwhich any part of it is contained within the body, i.e. the device maybe wholly or partly in-dwelling.

The surface can be made of any material. For example it may be metal,e.g. aluminum, steel, stainless steel, chrome, titanium, iron, alloysthereof, and the like. The surface can also be plastic, for example,polyolefin (e.g., polyethylene, (Ultra-High Molecular Weight)polyethylene, polypropylene, polystyrene, poly(meth)acrylate,acrylonitrile, butadiene, ABS, acrylonitrile butadiene, etc.), polyester(e.g., polyethylene terephthalate, etc.), and polyamide (e.g., nylon),combinations thereof, and the like. Other examples include acetalcopolymer, polyphenylsulfone, polysulfone, polythermide, polycarbonate,polyetheretherketone, polyvinylidene fluoride, poly(methyl methacrylate)and poly(tetrafluoroethylene). The surface can also be brick, tile,ceramic, porcelain, wood, vinyl, linoleum, or carpet, combinationsthereof, and the like. The surfaces can also be food, for example, beef,poultry, pork, vegetables, fruits, fish, shellfish, combinationsthereof, and the like. The “treatment” of any such surface (i.e. theapplication to any such surface of an alginate oligomer together with anantibiotic) to combat infection by an MDR bacterium is encompassed bythe present invention

In an infection by an MDR bacterium, which may be treated according tothe present invention, the bacterium may occur in or on a surface in asubject. Furthermore, outside the context of medical treatment, bacteriamay also occur on biotic surfaces. Thus the invention includes thetreatment of biotic surfaces. A biotic or animate surface may includeany surface or interface in or on an animal, plant or fungal body. Itmay accordingly be viewed as a “physiological” or “biological” surface.It may be any internal or external body surface, including of any tissueor organ, which, in the case of an animal body, may includehaematological or haematopoietic tissue (e.g. blood). Dead or dying(e.g. necrotic) or damaged (e.g. inflamed or disrupted or broken) tissueis particularly susceptible to bacterial contamination, and such tissueis encompassed by the term “animate” or “biotic”. The surface may be amucosal or non-mucosal surface.

Representative biotic surfaces include, but are not limited to, anysurface in the oral cavity (e.g. teeth, gingiva, gingival crevice,periodontal pocket) the reproductive tract (e.g. cervix, uterus,fallopian tubes), the peritoneum, middle ear, prostate, urinary tract,vascular intima, the eye i.e. ocular tissue (e.g. the conjunctiva,corneal tissue, lachrymal duct; lachrymal gland, eyelid) the respiratorytract, lung tissue (e.g. bronchial and alveolial), heart valves,gastrointestinal tract, skin, scalp, nails and the interior of wounds,particularly chronic wounds and surgical wounds, which may be topical orinternal wounds. Other surfaces include the exterior of organs,particularly those undergoing transplantation, for example, heart,lungs, kidney, liver, heart valve, pancreas, intestine, corneal tissue,arterial and venous grafts and skin.

In one aspect the surface will not be mucosal, or more particularly willnot have a hyperviscous mucus coating. The skilled person will be ableto determine when the mucus at a given surface is hyperviscous. In oneembodiment the surface will not be the surface of a mucus-secretingtissue. More particularly in such an embodiment the surface will not bethe surface of a mucus-coated tissue. The skilled person will know fromhis common general knowledge the tissues that secrete mucus and thosethat are mucus-coated.

The location may also be a location that is not a surface. In otherwords the bacterium can be found within an material as well as on itssurface. The material can be chemically heterogeneous as well aschemically homogenous. The material can also be constructed or formedfrom or comprise different parts or components. The material can be apart of a larger material or entity. The material may be or comprise thematerials from which the above mentioned surfaces are formed. In someinstances the material can be considered to be an object, which termscovers volumes of liquids wherever found. The material may comprise anyof the above described surfaces. The material may be abiotic or biotic(inanimate or animate) as is discussed above in relation to surfaces.For instance, the material might be, completely or in part, a solid, aliquid, a semi solid, a gel or a gel-sol. Thus, for example, thebacterium might be present in body fluids (e.g. blood, plasma, serum,cerebrospinal fluid, GI tract contents, semen, sputum and otherpulmonary secretions); tissues (e.g. adrenal, hepatic, renal,pancreatic, pituitary, thyroid, immune, ovarian, testicular, prostate,endometrial, ocular, mammary, adipose, epithelial, endothelial, neural,muscle, pulmonary, epidermis, osseous); cell and tissue culture media;cell and tissue cultures; clinical/scientific waste materials (which cancomprise any of the preceding materials); pharmaceuticals (e.g. tablets,pills, powders, lozenges, sachets, cachets, elixirs, suspensions,emulsions, solutions, syrups, aerosols, sprays, compositions for use innebulisers, ointments, soft and hard gelatine capsules, suppositories,sterile injectable solutions, sterile packaged powders); animal or humanfood stuffs (e.g. meat, fish, shellfish, vegetables, cereals, diaryproducts, fruit juices, vegetable juices, sauces, stocks, soups,confectionary, alcoholic beverages, condiments); personal hygieneproducts (e.g. toothpaste, mouthwash, shampoo, soap, deodorant, showergel); cosmetics (e.g. lip gloss, eye shadow, foundation); drinking watersupplies; waste water supplies; agricultural feedstuffs and watersupplies; insecticide, pesticide and herbicide formulations; industriallubricants and so on. Liquids, semi solids, gels or gel-sols are ofnote. The body fluids and tissues may be treated in vitro/ex vivo aswell as it being possible to treat the same in vivo.

In certain embodiments the bacterium will be in a biofilm. In otherembodiments the bacterium will not be in a biofilm. (e.g. will begrowing planktonically). Put differently, the bacterium will be, or willnot be, in a biofilm mode of growth; or will be, or will not be, in anon-biofilm mode of growth.

By “biofilm” it is meant a community of microorganisms characterized bya predominance of sessile cells that are attached to a substratum orinterface or to each other (some motile cells may also be present) andthat are embedded in a matrix of extracellular polymers (morespecifically extracellular polymers that they have produced)characterised in that the microorganisms of this colony exhibit analtered phenotype with respect to growth rate and gene transcription(for example as compared to their “non-biofilm” or free-floating orplanktonic counterparts). By “in a biofilm”, it is meant that thebacterium targeted by the method of the invention is within (completelyor in part), on or associated with the polymer matrix of a biofilm.Viewed differently, bacteria that are “not in a biofilm” are organismsthat are either in isolation, e.g. planktonic, or if in an aggregationof a plurality of organisms, that aggregation is unorganised and/or isdevoid of the matrix characteristic of a biofilm. In each case, theindividual bacteria do not exhibit an altered phenotype that is observedin their biofilm dwelling counterparts.

It is well appreciated that Acinetobacter organisms can form a capsulefrom extracellular polymers (e.g. polysaccharides) that they haveproduced and Acinetobacter organisms are typically found with such acapsule. It is also well appreciated that the simple presence of apolymer capsule of an Acinetobacter organism is not functionallyequivalent to a biofilm mode of growth and the presence of such acapsule is therefore not in itself indicative of a biofilm phenotype.Thus, it will also be appreciated that Acinetobacter organisms that are“not in a biofilm” may still be in contact a matrix of extracellularpolymers that they have produced (i.e. the capsule), but such organismswill not exhibit an altered phenotype that is observed in their biofilmdwelling counterparts. Thus, in the particular case of Acinetobacter, by“in a biofilm” it is meant that the Acinetobacter organism is within(completely or in part), on or associated with the polymer matrix of abiofilm and has an phenotype characteristic of Acinetobacter organismsin a biofilm (i.e. a phenotype that is altered with respect to growthrate and gene transcription, for example as compared to “non-biofilm” orfree-floating or planktonic Acinetobacter organisms. Acinetobacterorganisms that are “not in a biofilm” are organisms that are either inisolation, e.g. planktonic, or if in an aggregation of a plurality oforganisms, that aggregation is unorganised. In each case, the individualAcinetobacter organisms do not exhibit an altered phenotype that isobserved in their biofilm dwelling counterparts.

From the forgoing it is clear that the methods of the invention, i.e.those described above, have medical and non-medical applications. Inparticular, the invention provides a method for combating contaminationof a site with bacteria that are MDR, in particular the treatment in asubject of a bacterial infection that is MDR, and also a method ofcombating a population of MDR bacteria. Thus, the method may be an invitro or an in vivo method. As explained in more detail below,“combating” includes both the treatment of an existing contamination orinfection, and treatment to prevent a contamination or infection fromoccurring, i.e. both “therapeutic”/reactionary and prophylactictreatments.

Accordingly, in one aspect of the invention there is provided a methodfor the treatment or prevention of an infection of a subject by an MDRbacterium, said method comprising administering to said subject apharmaceutically effective amount of an alginate oligomer together witha pharmaceutically effective amount of at least one antibiotic to whichthe bacterium is resistant.

Thus the invention provides an alginate oligomer for use together with(or in combination or conjunction with) at least one antibiotic in thetreatment or prevention of an infection of a subject by an MDRbacterium, wherein the bacterium is resistant to the antibiotic.

The term “use together” should be construed as discussed above, althoughit is particularly meant that a pharmaceutically effective amount of thealginate oligomer is administered at the same or substantially the sametime as or prior to, or after, administering a pharmaceuticallyeffective amount of the antibiotic.

Alternatively put, the invention provides the use of an alginateoligomer for the manufacture of a medicament for use together with anantibiotic in the treatment or prevention of an infection of a subjectby an MDR bacterium, wherein the bacterium is resistant to theantibiotic.

As noted above, the medicament may further comprise the antibiotic, andsingle or separate compositions or formulations may be provided andused, as discussed above.

This aspect of the invention also provides the use of an alginateoligomer together with an antibiotic in the manufacture of a medicamentfor use in the treatment of an infection of a subject by an MDRbacterium, wherein the bacterium is resistant to the antibiotic.

Also provided according to this aspect of the invention is a productcontaining an alginate oligomer and an antibiotic as a combinedpreparation for separate, simultaneous or sequential use in thetreatment or prevention of an infection of a subject by an MDR multidrugresistant bacterium, wherein the bacterium is resistant to theantibiotic.

The MDR bacterium can be any species of bacteria, e.g. those discussedabove and mentioned as preferred, e.g. a Burkholderia organism, e.g.Burkholderia cepacia. The antibiotic can be any antibiotic e.g. thosediscussed above and mentioned as preferred, e.g. a macrolide, e.g.azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycinor spiramycin.

The subject may be any human or non-human animal subject, but moreparticularly may be a vertebrate, e.g. an animal selected from mammals,birds, amphibians, fish and reptiles. The animal may be a livestock or adomestic animal, or an animal of commercial value, including laboratoryanimals or an animal in a zoo or game park. Representative animalstherefore include dogs, cats, rabbits, mice, guinea pigs, hamsters,horses, pigs, sheep, goats, cows, chickens, turkeys, guinea fowl, ducks,geese, parrots, budgerigars, pigeons, salmon, trout, cod, haddock, seabass and carp. Veterinary uses of the invention are thus covered. Thesubject may be viewed as a patient. Preferably the subject is a human.

The term “in a subject” is used broadly herein to include sites orlocations inside a subject or on a subject, e.g. an external bodysurface, and may include in particular infection of a medical devicee.g. an implanted or “in-dwelling” medical device. The term “in apatient” should be interpreted consistently with this.

The location of the infection is not restricted and may be any of thesites or locations in a subject described above. Administering thealginate oligomer and the antibiotic to the subject preferably resultsin the infected location being contacted with an alginate oligomer andantibiotic in amounts sufficient to treat the infection.

The infection may be acute, or alternatively chronic, e.g. an infectionthat has persisted for at least 5 or at least 10 days, particularly atleast 20 days, more particularly at least 30 days, most particularly atleast 40 days.

In this aspect of the invention the infection may occur on a surface inor on the subject (i.e. a biotic surface as discussed above) and/or asurface of a medical device, particularly an implantable or“in-dwelling” medical device, representative examples of which arediscussed above.

In one embodiment the methods or uses of the invention may comprise astep in which the subject is identified (e.g. diagnosed) as having orsuspected to have an MDR bacterial infection or being a candidate thatis at risk of or susceptible to an MDR bacterial infection.

In particular embodiments the invention may provide for the treatment ofrespiratory infections, e.g. cystic fibrosis, pneumonia, COPD, COAD,COAP, bacteraemia, septicaemia, septic shock, sepsis, meningitis, orpoisoning by bacterially derived toxins.

An MDR bacterial infection can occur in any subject but some subjectswill be more susceptible to infection that others. Subjects who aresusceptible to MDR bacterial infection include, but are not limited to,subjects whose epithelial and/or endothelial barrier is weakened orcompromised, subjects whose secretion-based defences to microbialinfection have been abrogated, disrupted, weakened or undermined, andsubjects who are immunocompromised, immunodeficient or immunosuppressed(i.e. a subject in whom any part of the immune system is not workingnormally, or is working sub-normally, in other words in whom any part ofthe immune response, or an immune activity is reduced or impaired,whether due to disease or clinical intervention or other treatment, orin any way).

Representative examples of subjects who are susceptible to MDR bacterialinfection include, but are not limited to, subjects with apre-established infection (e.g. with bacteria, viruses, fungi orparasites such as protozoa), especially subjects with HIV, subjects withbacteraemia, sepsis and subjects with septic shock; subjects withimmunodeficiency, e.g. subjects preparing for, undergoing or recoveringfrom chemotherapy and/or radiotherapy, organ (e.g. bone marrow, liver,lung, heart, heart valve, kidney, etc.) transplant subjects (includingautograft, allograft and xenograft patients); subjects with AIDS;subjects resident in a healthcare institution, e.g. hospital, especiallysubjects in intensive care or critical care (i.e. those units concernedwith the provision of life support or organ support systems topatients); subjects on respiratory ventilators; subjects suffering fromtrauma; subjects with burns, subjects with acute and/or chronic wounds;neonatal subjects; elderly subjects; subjects with cancer (definedbroadly herein to include any neoplastic condition; malignant ornon-malignant), especially those with cancers of the immune system (e.g.leukaemias, lymphomas and other haematological cancers); subjectssuffering from auto-immune conditions such as rheumatoid arthritis,diabetes mellitus type I, Crohn's disease, especially those undergoingimmunosuppression treatment for those diseases; subjects with reduced orabrogated epithelial or endothelial secretion (e.g. mucous, tears,saliva) and/or secretion clearance (e.g. subjects with poorlyfunctioning cilia on mucosal tissue and/or patients with hyperviscousmucous (e.g. smokers and subjects with COPD, COAD, COAP, bronchitis,cystic fibrosis, emphysema, lung cancer, asthma, pneumonia orsinusitis)) and subjects fitted with a medical device.

MDR bacteria are commonly encountered in healthcare institutions due inpart to the close proximity of subjects with bacterial infection's andthe widespread-use of antibiotics. MDR bacteria, e.g. from the generaPseudomonas, Klebsiella, Burkholderia, Providencia and Acinetobacter,are therefore often involved in nosocomial infections and accordinglythe invention can be seen as providing treatments for MDR nosocomialinfections.

Thus, subjects in whom MDR infections may particularly be combatedaccording to the present invention include patients who are impaired,whether due to poor perfusion, repetitive trauma, poor nutrition, pooroxygenation or white cell dysfunction.

Of particular note are subjects that have undergone physical trauma. Thetrauma itself might cause a weakening in or compromisation of anepithelial and/or endothelial barrier of the subject or the subject maybecome immunocompromised in response to the trauma (a shock response).The term “trauma” refers broadly to cellular attack by foreign bodiesand/or physical injury of cells. Included among foreign bodies aremicroorganisms, particulate matter, chemical agents, and the like.Included among physical injuries are mechanical injuries; thermalinjuries, such as those resulting from excessive heat or cold;electrical injuries, such as those caused by contact with sources ofelectrical potential; and radiation damage caused, for example, byprolonged, extensive exposure to infrared, ultraviolet or ionizingradiations.

Also of particular note are subjects that have a burn. Any burn, inparticular a severe burn, has a significant impact on the integrity ofthe epithelial and/or endothelial barrier of the subject and the subjectwill often become immunocompromised in response to the burn (a shockresponse).

Typical burn-causing agents are extremes of temperature (e.g. fire andliquids and gases at extreme temperature), electricity, corrosivechemicals, friction and radiation. The extent and duration of exposure,together with the intensity/strength of the agent, result in burns ofvarying severity. Scalding (i.e. trauma associated with high temperatureliquids and/or gases) is considered to be a burn.

Epidermal burn severity is commonly classified in two ways. Most commonis the classification by degree. First-degree burns are usually limitedto erythema (redness) in the general area of the injury and a whiteplaque at the site of injury. The cellular trauma of these burns extendsonly as deep as the epidermis. Second-degree burns also display erythemain the general area of the injury but with superficial blistering of theepidermis. The cellular trauma of second-degree burns involves thesuperficial (papillary) dermis and may also involve the deep (reticular)dermis layer. Third-degree burns are those in which the epidermis islost with damage to the hypodermis. Damage is typically extremeincluding charring. Sometimes eschar, (dry, black necrotic tissue) willbe present. Third-degree burns may require grafting. In fourth-degreeburns catastrophic damage of the hypodermis occurs, e.g. the hypodermisis completed lost, with damage extending to the underlying muscle,tendon, and ligament tissue. Charring and eschar are observed. Graftingis required if the burn does not prove to be fatal.

Another common classification system is the classification by thickness.“Superficial thickness” burns correspond to first degree burns. Thespectrum of second degree burns is covered by two classes of “partialthickness” burns. “Partial thickness-superficial” are burns that affectthe epidermis only as far as the papillary dermis. “Partialthickness-deep” are burns that affect the dermis as far as the reticulardermis. “Full thickness” burns correspond to third and fourth degreeburns.

Some physical injuries, e.g. some burns, and cellular attacks by foreignbodies result in the formation of a wound. More specifically a wound maybe considered to be a breach in, or denudement of, a tissue. Wounds mayalso be caused by a spontaneously forming lesion such as a skin ulcer(e.g. a venous, diabetic or pressure ulcer), an anal fissure or a mouthulcer.

Wounds are typically defined as either acute or chronic. Acute woundsare wounds that proceed orderly through the three recognised stages ofthe healing process (i.e. the inflammatory stage, the proliferativestage and the remodelling phase) without a protracted timecourse.Chronic wounds, however, are those wounds that do not complete theordered sequence of biochemical events of the healing process becausethe wound has stalled in one of the healing stages. Commonly, chronicwounds are stalled in the inflammatory phase. In accordance with aparticular aspect of the present invention, a chronic wound is a woundthat has not healed within at least 40 days, particularly at least 50days, more particularly at least 60 days, most particularly at least 70days.

As discussed above, wounds are an ideal environment for an MDR bacterialinfection, particularly chronic infection, due to their lack of anepithelial barrier and the availability of substrate and surface formicrobial attachment and colonisation. Problematically, infection of awound often delays healing further and thus renders that wound moresusceptible to established infection. The methods of the invention aretherefore effective in the treatment and prevention of MDR bacterialinfection of wounds and the use of the methods of the invention in thetreatment of wounds, especially chronic wounds, represents one preferredaspect of the present invention.

Therefore, in an embodiment of the invention there is provided analginate oligomer for use together with (or in combination orconjunction with) an antibiotic in the treatment or prevention of theinfection of a subject by an MDR bacterium, wherein the bacterium isresistant to the antibiotic, particularly chronic infection by an MDRbacterium in the above-mentioned subjects, in particular in subjectswith respiratory diseases or disorders e.g. cystic fibrosis, COPD, COAD,COAP, pneumonia, wounds, burns and/or traumas.

Through the ability to treat and prevent infection of wounds by an MDRbacterium the alginate oligomers and antibiotics of the invention asdefined herein can remove one of the obstacles to wound healing andtherefore the alginate oligomers and antibiotics defined above are alsoeffective in the promotion of healing of acute and chronic woundsinfected with or at risk of infection with an MDR bacterium which isresistant to any of said antibiotics

By promotion of healing it is meant that the treatment accelerates thehealing process of the wound in question (i.e. the progression of thewound through the three recognised stages of the healing process). Theacceleration of the healing process may manifest as an increase in therate of progression through one, two or all of the healing stages (i.e.the inflammatory stage, the proliferative stage and/or the remodellingphase). If the wound is a chronic wound that is stalled in one of thehealing stages the acceleration might manifest as the restarting of thelinear, sequential healing process after the stall. In other words, thetreatment shifts the wound from a non-healing state to a state where thewound begins to progress through the healing stages. That progressionafter the restart may be at a normal rate or even a slower rate comparedwith the rate a normal acute wound would heal.

The alginate oligomers and antibiotics of the invention may be usedtogether (or in combination or conjunction) to treat or prevent MDRbacterial infections wherever they may occur in or on the body. Thus, inanother embodiment, the infection may be an infection of a medicaldevice by an MDR bacterium, particularly an in-dwelling medical device,e.g. endotracheal and tracheostomy tubes.

The alginate oligomers and antibiotics of the invention may be usedtogether (or in combination or conjunction) as oral healthcare agents,for example in the control of dental plaque, e.g. to reduce it or toprevent, reduce or delay its development by inhibiting growth of MDRplaque bacteria on teeth or dental/oral prostheses. The alginateoligomers and antibiotics of the invention may also be used together (orin combination or conjunction) in the treatment and prevention of MDRinfections or MDR infectious disease which may occur in the oral cavity,for example gingivitis and periodontitis

Conveniently, the alginate oligomers and/or antibiotics can be appliedby any oral health/oral hygiene delivery system. This may be through theuse of toothpastes, dental gels, dental foams and mouthwashes. Removabledentures and other removable dental prostheses may be treated outside ofthe oral cavity with the same compositions or other suitablepharmaceutically acceptable compositions. The alginate oligomers and/orantibiotics can also be incorporated into compositions that are appliedto the oral cavity (or applied to removable dentures and other removabledental prostheses outside of the oral cavity) to form a coating thatpersists on surfaces over time, or that releases the alginate oligomersand/or antibiotics from the coated surfaces over time, and which inhibitthe growth of MDR bacteria in the oral cavity and on the surfaces ofremovable dentures and other removable dental prostheses.

Whilst the treatment of MDR bacterial infections of the lungs andrespiratory tract and all areas of the body is generally covered by thepresent invention, in one embodiment, the medical uses of the inventionare not directed to the treatment of (i) infections in the respiratorytract, e.g. in patients suffering from COPD's (chronic obstructivepulmonary diseases), in particular the sinuses and the lungs, inparticular in the treatment of cystic fibrosis, chronic obstructivepulmonary disease, emphysema, bronchitis and sinusitis; (ii) in themiddle ear of patients suffering from glue ear; or (iii) in thereproductive tract of female patients with impaired fertility; or (iv)in the digestive tract of patients with digestive tract malfunction(e.g. constipation).

In specific embodiments of the invention the alginate oligomers andantibiotics of the invention may be used together (or in combination orconjunction) in the treatment or prevention of native valveendocarditis, acute otitis media, chronic bacterial prostatitis,pneumonia (in particular ventilator associated pneumonia) associatedwith MDR bacteria; respiratory diseases associated with MDR bacteria(which may include COPD, COAD, COAP, pneumonia, cystic fibrosis andasthma); and device related MDR bacterial infections associated withimplantable or prosthetic medical devices (e.g. prosthetic valveendocarditis or the infection of lines or catheters or artificial jointsor tissue replacements or endotracheal or tracheotomy tubes).

In further embodiments the alginate oligomers and antibiotics of theinvention are used together to control MDR infections in the eye, e.g.to reduce them, or prevent, reduce or delay their development. Inparticular, the alginate and antibiotics of the invention are usedtogether to treat or prevent MDR bacterial conjunctivitis and theresultant keratoconjunctivitis sicca (also known as dry eye) that canresult through the blockage of the lachrymal gland.

As mentioned previously, in certain embodiments, the above MDR bacterialinfections and associated conditions are, or involve, biofilm, in otherwords they are biofilm infections. In other embodiments the above MDRbacterial infections and associated conditions are not, or do notinvolve biofilm.

In a further aspect the invention provides a method for combatingcontamination of a site with MDR bacteria, said method comprisingcontacting the site and/or the MDR bacteria with (an effective amountof) an alginate oligomer together with (an effective amount of) at leastone antibiotic to which the bacteria are resistant. Such a method mayparticularly be an in vitro method, and the site may be any surface orlocation discussed above.

“Combating contamination” includes both preventative and reactionarymeasures or treatments and therefore covers the prevention as well asthe reduction, limitation, or elimination of contamination.

By “contamination” it is meant the unwanted presence of a bacterium(e.g. an MDR bacterium) at a particular site or location. Contaminationcan be considered to cover colonisation of a location by a bacterium(e.g. an MDR bacterium), i.e. the establishment of a bacterium (e.g. anMDR bacterium) at a location and the expansion of the numbers of thatorganism by replication or the recruitment of additional bacteria, whichmay be of the same or of a different type. In one embodiment thecolonisation process will not involve the formation of a biofilm.

The site or location of the contamination or potential contamination isnot restricted and can be any of the various sites or locationsdescribed or mentioned above, e.g. it can be in vitro or in vivo, butparticularly in this aspect of the invention it will be an “in vitro” or“ex vivo” site or location (e.g. an inanimate or abiotic site orlocation). However, the site or location may be in a subject and inwhich case a pharmaceutically effective amounts of the alginate oligomerand the antibiotic are administered to the subject.

In one particular embodiment the various aspects of the invention can beapplied to the decontamination of clinical, scientific and industrialwaste materials. In another particular embodiment the various aspects ofthe invention can be used to decontaminant transplant tissue (e.g.heart, lungs, kidney, liver, heart valve, pancreas, intestine, cornealtissue, arterial and venous grafts and skin) and medical devices (e.g.endotracheal and tracheostomy tubes) prior to implantation. In anotherembodiment the various aspects of the invention can be considered tocover the use of alginate oligomers together with antibiotics asanti-MDR bacterial preservative agents in materials, especiallysolutions and liquids.

In another embodiment, the methods of the invention may further comprisea step in which the bacteria being targeted will be determined as being,or alternatively not being in, or involving, a biofilm.

In other embodiments of the methods of the invention the methods maycomprise a step in which it is determined (e.g. ascertained oridentified) that the bacterium is resistant to a particularantibiotic(s). In a step in place of, or in addition to, the previouslydescribed step, there may be a step in which it is determined that thebacterium is an MDR bacterium. Any convenient test can be used here, forinstance those described above, or any technique for identifying knownand characterised bacteria (e.g. bacteria already identified as beingantibiotic and/or multidrug resistant). In a further step it may beascertained whether or not a particular resistance is acquired orintrinsic, e.g. by comparison to typical or wild type bacteria of thesame species.

In any of the aspects, uses or methods of the invention the MDR bacteriaand the antibiotic can be any of the bacteria and antibiotics definedabove and especially any, or combinations thereof, stated as preferred.For example, the MDR bacteria may be a bacteria from an MDR strain ofbacteria. Also for example, the MDR bacteria may be a Burkholderiaorganism, e.g. Burkholderia cepacia. Also for example the antibiotic maybe a macrolide, e.g. azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin or spiramycin.

The term “contacting” encompasses any means of delivering the alginateoligomer and the antibiotic to the MDR bacterium, whether directly orindirectly, and thus any means of applying the alginate oligomer and theantibiotic to the MDR bacterium or exposing the MDR bacterium to thealginate oligomer and the antibiotic e.g. applying the alginate oligomerand the antibiotic directly to the MDR bacterium or administering thealginate oligomer and the antibiotic to a subject within which or onwhich the MDR bacterium is present, e.g. a subject infected with an MDRbacterium.

More particularly the MDR bacterium will be contacted with an effectiveamount of the alginate oligomer and the antibiotic, more particularly anamount of the alginate oligomer and an amount of the antibiotic thattogether (or in combination or conjunction) overcome the resistance ofthe MDR bacterium to the antibiotic and therefore inhibit the viabilityand/or growth of the MDR bacterium and therefore treat or prevent theinfection/contamination.

An “effective amount” of the alginate oligomer and the antibiotic isthat amount of alginate oligomer and that amount of the antibiotic thattogether (or in combination or conjunction) provide measurable reductionin the resistance (or measurable increase in susceptibility ormeasurable decrease in tolerance) to the antibiotic displayed by thebacterium (e.g. using the above-described indicators of resistance). Incertain embodiments the “effective amount” of the alginate oligomer andthe antibiotic is that amount of alginate oligomer and that amount ofthe antibiotic that together (or in combination or conjunction) providemeasurable inhibition of the growth of an MDR bacterium, or populationthereof, that is being targeted, e.g. which is resistant to theantibiotic.

A “pharmaceutically effective” amount of the alginate oligomer and theantibiotic is that amount of alginate oligomer and that amount of theantibiotic that together (or in combination or conjunction) provide ameasurable reduction in the resistance (or measurable increase insusceptibility or measurable decrease in tolerance) to the antibioticdisplayed by the MDR bacterium (e.g. using the above-describedindicators of resistance) in a subject and/or a measurable treatment orprevention of the infection by an MDR bacterium that is being targeted.

The skilled man would easily be able to determine what aneffective/pharmaceutically effective amount of alginate oligomer andantibiotic would be on the basis of routine dose response protocols and,conveniently, the routine techniques for assessing microbial growthinhibition etc., as discussed below. The skilled man would, withoutundue burden, also be able to optimise these amounts to maximise thecombinatorial effects of the alginate oligomer and antibiotic in histarget system.

By “growth of an MDR bacterium” it is meant both an increase in the sizeof an MDR bacterium or in the amount and/or volume of the constituentsof an MDR bacterium (e.g. the amount of nucleic acid, the amount ofprotein, the number of nuclei, the numbers or size of organelles, thevolume of cytoplasm) and an increase in the numbers of the MDRbacterium, i.e. an increase in the replication of the MDR bacterium.

Typically growth of an MDR bacterium is accompanied by the enlargementof the organism. The growth of MDR bacteria can be measured with routinetechniques. For instance, microscopic examination of microorganismmorphology over time, or assays to measure changes in the quantities ofprotein or nucleic acid (e.g. DNA) in general, or the changes in thequantities of specific proteins or nucleic acids, can be used. Theskilled man would easily be able to select suitable markers to follow.Conveniently, so called housekeeping genes (e.g. β-actin, GAPDH(glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinatedehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L(HBS1-like protein), AHSP (alphahaemoglobin stabilising protein), andβ2M (beta-2-microglobulin)), 16S RNA and virus genes, and theirexpression products can be monitored.

By “replication of an MDR bacterium” or “replication of a bacterium” itis meant the act by which the (MDR) bacterium reproduces. Typically thisis by binary fission where a microorganism divides into two. To supportthe division of the microorganism into two, binary fission is normallypreceded by enlargement of the dividing microorganism and an increase inthe amount and/or volume of cellular constituents. Replication resultsin an increase in the number of cells and so may be followed by anymethod of assessing microorganism numbers in a population. Anotheroption is to follow the process in real time by visual examination witha microscope. The time it takes for microorganism to replicate (i.e.produce another version of itself) is the generation time. Generationtime will depend on the conditions in which the (MDR) bacterium isfound. The rate of replication can be expressed in terms of thegeneration time.

By “inhibiting the growth of an MDR bacterium” or inhibiting the growthof a bacterium” it is meant that measurable growth (e.g. replication) ofan (MDR) bacterium, or the rate thereof, is reduced. Preferablymeasurable growth (e.g. replication) of an (MDR) bacterium, or the ratethereof, is reduced by at least 50%, more preferably at least 60%, 70%,80% or 90%, e.g. at least 95%. Preferably, measurable growth (e.g.replication) is ceased. Growth in terms of microbial size increase orexpansion etc. may be inhibited independently of replication and viceversa.

Suitable doses of alginate oligomer and antibiotic will vary fromsubject to subject and can be determined by the physician or veterinarypractitioner in accordance with the weight, age and sex of the subject,the severity of the condition, the mode of administration and also theparticular alginate oligomer or antibiotic selected. Typically thealginate oligomers of the invention will be applied to the locationundergoing treatment at a local concentration of at least 0.5%,preferably at least 2% or at least 4%, more preferably at least 6% andmost preferably at least 10% weight by volume. Typically the antibioticof the invention will be applied to the location undergoing treatment ata local concentration of at least 1 μg/ml, preferably at least 4, atleast 8, at least 16, at least 32, at least 64, at least 128, at least256 or at least 512, 1024, 2048 or 4096 μg/ml.

“Treatment” when used in relation to the treatment of a medicalcondition/infection in a subject in accordance with the invention isused broadly herein to include any therapeutic effect, i.e. anybeneficial effect on the condition or in relation to the infection.Thus, not only included is eradication or elimination of the infection,or cure of the subject or infection, but also an improvement in theinfection or condition of the subject. Thus included for example, is animprovement in any symptom or sign of the infection or condition, or inany clinically accepted indicator of the infection/condition (forexample a decrease in wound size or an acceleration of healing time).Treatment thus includes both curative and palliative therapy, e.g. of apre-existing or diagnosed infection/condition, i.e. a reactionarytreatment.

“Prevention” as used herein refers to any prophylactic or preventativeeffect. It thus includes delaying, limiting, reducing or preventing thecondition (which reference includes infection and contamination, asapplicable, in the different aspects of the invention) or the onset ofthe condition, or one or more symptoms or indications thereof, forexample relative to the condition or symptom or indication prior to theprophylactic treatment. Prophylaxis thus explicitly includes bothabsolute prevention of occurrence or development of the condition, orsymptom or indication thereof, and any delay in the onset or developmentof the condition or symptom or indication, or reduction or limitation onthe development or progression of the condition or symptom orindication.

Specifically, the alginate oligomers and antibiotics of the inventioncan be taken together (or in combination or conjunction) as aprophylactic treatment, for example to prevent, or at least minimise therisk, of infection or contamination by an MDR bacterium resistant to theantibiotic.

The aspect of the invention concerning the combating (treatment orprevention) of infection by an MDR bacterium is of particular utility inthe care of hospitalised patients as the risk of contracting annosocomial infection (commonly known as hospital related/acquiredinfection or healthcare-associated infection) by an MDR bacterium can beminimised with a prophylactic regime of the alginate oligomers andantibiotics defined herein. This aspect of the invention is also ofparticular utility in the care of subjects suffering from trauma,subjects with a burn and subjects with wounds, all of which, asdiscussed above, are more susceptible to infection by MDR bacteria thana subject that is not affected similarly.

Generally, subjects in need of treatment or prophylaxis according to theinvention will be diagnosed as suffering or at risk from infection by anMDR bacterium, e.g. identified as having or at risk of developing aninfection by an MDR bacterium.

Specifically, the alginate oligomers and antibiotics of the inventioncan be taken together (or in combination or conjunction) as aprophylactic treatment to prevent, or at least minimise the risk, ofdeveloping an infection by an MDR bacterium resistant to the chosenantibiotic(s), including for example the infection of wounds by an MDRbacterium; native valve endocarditis, acute otitis media, chronicbacterial prostatitis, associated with an MDR bacterium; infections ofthe respiratory tract and lungs by an MDR bacterium (e.g. cysticfibrosis, COPD, COAD, COAP, pneumonia, or other respiratory diseases) orinfection of a medical (e.g. in-dwelling) medical device by an MDRbacterium.

The invention encompasses the use of a single alginate oligomer or amixture (multiplicity/plurality) of different alginate oligomers. Thus,for example, a combination of different alginate oligomers (e.g. two ormore) may be used.

The invention encompasses the use of a single antibiotic or a mixture(multiplicity/plurality) of different antibiotics. Thus, for example, acombination of different antibiotics (e.g. two or more) may be used. TheMDR bacterium may be sensitive to the further antibiotic(s) used or maybe resistant to the further antibiotic(s) used.

In one advantageous embodiment of the invention the alginate oligomersand antibiotic may be used in the methods of the invention inconjunction or combination with a further anti-microbial agent(hereinafter “further anti-microbial agent”)

In the context of a medical use, such an anti-microbial agent may be anyclinically-useful anti-microbial agent and particularly an antibiotic oran antiviral or antifungal agent. In the context of non-clinical uses,the anti-microbial agent may again be any anti-microbial agent used forsuch purposes, e.g. any disinfectant or antiseptic or cleaning orsterilising agent. The agents may be used separately, or together in thesame composition, simultaneously or sequentially or separately, e.g. atany desired time interval.

Thus, by way of representative example, the further anti-microbial agentmay be used after the alginate oligomer and/or the antibiotic, but apreceding or simultaneous or intervening use may be beneficial in somecircumstances.

The choice of anti-microbial agent will of course need to be appropriatefor the location undergoing treatment, but for instance anti-microbialagents, e.g. antibiotics, antifungals, antivirals, antiseptics may beused and/or sterilising conditions such as irradiation (e.g. UV, X-ray,gamma) extremes of temperature, and extremes of pH.

Representative antibiotics include those listed above, especially thosestated as preferred.

Representative antiseptics include, but are not limited to chlorinebleach (sodium hypochlorite), quaternary ammonium compounds (e.g.benzalkonium chloride, cetyl trimethylammonium bromide; cetylpyridiniumchloride), hydrogen peroxide, phenol compounds (e.g. TCP), alcohols(e.g. ethanol), Virkon™, iodine compounds (e.g. povidone-iodine), silvercompounds (e.g. elemental silver nano/microparticles).

Antimicrobial surfactants are another class of antiseptics. These arecompounds that disrupt microbial cell membranes and other structuralcomponents and therefore inhibit growth and/or viability ofmicroorganisms. Antimicrobial surfactants and their use in antimicrobialcompositions is well known in the art should further guidance be neededthe discussion of antimicrobial surfactants in “Preservative-free andself-preserving cosmetics and drugs—Principles and practice”, Ed. Kabaraand Orth, Marcel Dekker, N.Y., N.Y., 1997, is explicitly incorporated byreference in its entirety. Antimicrobial surfactants may be anionic,cationic, non-ionic or amphoteric. Examples of antimicrobial anionicsurfactants include, but are not limited to, sodium dodecyl sulfate(sodium lauryl sulfate), sodium dodecyl aminopropionic acid, sodiumricinoleate, bile acids, alkylaryl sulfonates, Grillosan DS7911,disodium undecylenic acid monoethanol amidosulfosuccinate. Examples ofantimicrobial cationic surfactants include, but are not limited to, thequaternary ammionium compounds, the aminimides and chlorhexidinecompounds. Examples of antimicrobial non-ionic surfactants include, butare not limited to, the monoesters of fatty acids,polyethyleneglycomonoesters of alkyldihydroxybenzoic acids, glucosaminederivatives and diethanolamides of N-lauroyl dipeptides. Examples ofantimicrobial amphoteric surfactants include, but are not limited to,the alkyl betaines, the alkylamidopropylbetaines, the alkylaminopropionates, the alkyliminodipropionates and the alkylimidazolines.

Representative antifungals include, but are not limited to the polyenes(e.g. natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin;the imidazoles (e.g. miconazole, ketoconazole, clotrimazole, econazole,bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole,sertaconazole, sulconazole, tioconazole); the triazoles (e.g.fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole,voriconazole, terconazole); the allylamines (e.g. terbinafine,amorolfine, naftifine, butenafine); and the echinocandins (e.g.anidulafungin, caspofungin, micafungin).

Representative antivirals include, but are not limited to abacavir,acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir,atripla, boceprevir, cidofovir, combivir, darunavir, delavirdine,didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide,entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet,ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, indinavir,inosine, interferon type III, interferon type, II interferon type I,lamivudine, lopinavir, loviride, maraviroc, moroxydine, nelfinavir,nevirapine, nexavir, oseltamivir, penciclovir, peramivir, pleconaril,podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir,saquinavir, stavudine, tenofovir, tenofovir disoproxil, tipranavir,trifluridine, trizivir, tromantadine, truvada, valaciclovir,valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine,zanamivir, and zidovudine.

The further anti-microbial agent may conveniently be applied before,simultaneously with, following or between the alginate oligomer and/orthe antibiotic. Conveniently the further anti-microbial agent is appliedat substantially the same time as the alginate oligomer and/or theantibiotic or afterwards. For example, the further anti-microbial agentis applied at least 1 hour, preferably at least 3 hours, more preferablyat least 5 and most preferably at least 6 hours after the alginateoligomer and/or the antibiotic is administered. In other embodiments thefurther antimicrobial may conveniently be applied or administered beforethe alginate oligomer and/or the antibiotic r, e.g. at least 1 hour, atleast 3 hours, at least 6 hours before the alginate oligomer and/or theantibiotic. In these embodiments the alginate oligomer and/or theantibiotic can be applied or administered with or without a furtherapplication of the further antimicrobial. To optimise the anti-microbialeffect of the further anti-microbial agent it can be given (e.g.administered or delivered) repeatedly at time points appropriate for theagent used. The skilled person is able to devise a suitable dosage orusage regimen. In long term treatments the alginate oligomer and/or theantibiotic can also be used repeatedly. The alginate oligomer can beapplied as frequently as the antibiotic and/or the furtheranti-microbial agent, but will typically be less frequently. Thefrequency required will depend on the location of the MDR bacterium,colony composition and the anti-microbial used and the skilled person isable to optimise the dosage or usage patterns to optimise results.

In an advantageous embodiment the alginate oligomer and/or theantibiotic may be used or applied after physical removal or reduction(e.g. debridement) of the colony/population comprising the MDR bacteriumcausing the infection at the location undergoing treatment.

Following removal of, or an attempt to remove, the colony/populationcomprising the MDR bacterium, the location may be contacted with thealginate oligomer for between 0 and 24 hours, particularly 2 and 12hours, more particularly 4 and 8 hours, most particularly 5 and 7 hours,e.g. 6 hours. Following this, the antibiotic, and if desired the furtheranti-microbial agent, may be applied. Such a scenario may be desirableor particularly applicable in a clinical setting. In the case of woundsinfected by an MDR bacterium, the duration of incubation can beconveniently be designed to correspond to scheduled changes of the wounddressing.

Physical removal of the colony/population comprising the MDR bacteriumcan be carried out with any suitable surgical, mechanical or chemicalmeans. Conveniently this can be the use of a liquid, gel, gel-sol,semi-solid compositions or gas applied at pressure to thecolony/population, sonication, laser, or by abrasive implement. Acomposition used in the removal itself or as a wash solution before,during or afterwards may conveniently contain the alginate oligomerand/or the antibiotic.

Accordingly, in one specific embodiment there is provided a debridementor wash composition e.g. solution for wounds containing an alginateoligomer, particularly any alginate oligomer as herein defined, and/oran antibiotic, particularly any antibiotic as herein defined (e.g. amacrolide, preferably selected from azithromycin, clarithromycin,dirithromycin, erythromycin, roxithromycin or spiramycin), for use inthe treatments and methods of the invention. Such a debridementcomposition will typically be a sterile solution, particularly anaqueous sterile solution or an oil-based sterile solution, and mayadditionally contain proteolysis enzymes (e.g. collagenase, trypsin,pepsin, elastase), an abrasive solid phase (e.g. colloidal silica,ground pumice, ground plant or animal shell).

Use of the alginate oligomers and the antibiotic in combination orconjunction with immunostimulatory agents may also be beneficial in theapplication of the methods of the invention in a clinical situation.These immunostimulatory agents may conveniently be used at timepointscorresponding to those described above in relation to anti-microbialagents and may optionally be used in combination with an alginateoligomer and/or the antibiotic and/or a further anti-microbial agentSuitable immunostimulatory agents include, but are not limited tocytokines e.g. TNF, IL-1, IL-6, IL-8 and immunostimulatory alginates,such as high M-content alginates as described for example in U.S. Pat.No. 5,169,840, WO91/11205 and WO03/045402 which are explicitlyincorporated by reference herein in their entirety, but including anyalginate with immunostimulatory properties.

Use of the alginate oligomers and the antibiotic in combination orconjunction with growth factors, e.g. PDGF, FGF, EGF, TGF, hGF andenzymes may also be beneficial in the medical uses of the invention.Representative examples of suitable enzymes include but are not limitedto proteases, e.g. serine proteases, metalloproteases and cysteineproteases (examples of these types of proteases are listed in EP0590746,the entire contents of which are incorporated herein by reference);nucleases, e.g. DNase I and II, RNase A, H, I, II, III, P, PhyM, R;lipases and enzymes capable of degrading polysaccharides.

Use of the alginate oligomers and the antibiotic in combination orconjunction with a physiologically tolerable mucosal viscosity reducingagent could also be beneficial, e.g. a nucleic acid cleaving enzyme(e.g. a DNase such as DNase I), gelsolin, a thiol reducing agent, anacetylcysteine, sodium chloride, an uncharged low molecular weightpolysaccharide (e.g. dextran), arginine (or other nitric oxideprecursors or synthesis stimulators), or an anionic polyamino acid (e.g.poly ASP or poly GLU). Ambroxol, romhexine, carbocisteine, domiodol,eprazinone, erdosteine, letosteine, mesna, neltenexine, sobrerol,stepronin, tiopronin are specific mucolytics of note.

Use of the alginate oligomers and the antibiotic in combination orconjunction with alpha blockers may also be beneficial in the medicaluses of the invention, in the treatment of chronic bacterial prostatitisespecially. Representative examples of suitable alpha blockers includebut are not limited to the selective alpha-1 blockers (e.g. doxazosin,dilodosin, prazosin, tamsulosin, alfuzosin, terazosin), and thenon-selective adrenergic blockers (e.g. phenoxybenzamine, phentolamine).

Use of the alginate oligomers and the antibiotic in combination orconjunction with bronchodilators may also be beneficial in the medicaluses of the invention, in the treatment of respiratory diseasesassociated with MDR bacteria especially (which may include COPD, COAD,COAP, pneumonia, cystic fibrosis, emphysema and asthma). Representativeexamples of suitable bronchodilators include but are not limited to theβ2 agonists (e.g. pirbuterol, epinephrine, salbutamol, salmeterol,levosalbutamol, clenbuterol), the anticholinergics (e.g. ipratropium,oxitropium, tiotropium) and theophylline.

Use of the alginate oligomers and the antibiotic in combination orconjunction with corticosteroids may also be beneficial in the medicaluses of the invention, in the treatment of respiratory diseasesassociated with MDR bacteria especially (which may include COPD, COAD,COAP, pneumonia, cystic fibrosis, emphysema and asthma). Representativeexamples of suitable corticosteroids include but are not limited toprednisone, flunisolide, triamcinolone, fluticasone, budesonide,mometasone, beclomethasone, amcinonide, budesonide, desonide,fluocinonide, fluocinolone, halcinonide. hydrocortisone, cortisone,tixocortol, prednisolone, methylprednisolone, prednisone, betamethasone,dexamethasone, fluocortolone, aclometasone, prednicarbate, clobetasone,clobetasol, and fluprednidene.

The alginate oligomers and the antibiotic can be used optionally withany other therapeutically active agent it may be desired to use, e.g. ananti-microbial agent, an anti-inflammatory agent (e.g. ananti-inflammatory steroid), an immunostimulatory agent, a mucosalviscosity reducing agent, a growth inhibitor or an enzyme or an alphablocker, a bronchodilator or a corticosteroid. The combined use of analginate oligomer and an antibiotic with a further therapeuticallyactive agent (e.g. an anti-microbial or anti-inflammatory agent, animmunostimulatory agent, a mucosal viscosity reducing agent, a growthinhibitor or an enzyme or an alpha blocker, a bronchodilator or acorticosteroid) may improve the clinical effects of the active agent andthis may advantageously allow the dose (e.g. the usual or normal dose)of the further therapeutically active agent to be reduced e.g. it may beused at its normal or usual dose or at a lower dose, for example at upto 50% (or at 50%) of its normal dose.

In the case of medical use, the alginate oligomers and antibiotics ofthe invention may be administered to the subject in any convenient formor by any convenient means, e.g. by topical, oral, parenteral, enteral,parenteral routes or by inhalation. Preferably the alginate andantibiotics will be administered by topical, oral or parenteral routesor by inhalation. The alginate oligomers and antibiotics need not be inthe same composition and need not be administered via the same route.

The skilled man will be able to formulate the alginate oligomers and theantibiotics of the invention into pharmaceutical compositions that areadapted for these routes of administration according to any of theconventional methods known in the art and widely described in theliterature.

The present invention therefore also provides a pharmaceuticalcomposition for use in any of the above-mentioned methods or usescomprising an alginate oligomer as defined herein together with at leastone pharmaceutically acceptable carrier, diluent or excipient. Thiscomposition may also comprise an antibiotic as defined herein.

The present invention therefore also provides a pharmaceuticalcomposition for use in any of the above-mentioned methods or usescomprising an antibiotic as defined herein together with at least onepharmaceutically acceptable carrier, diluent or excipient. Thiscomposition may also comprise an alginate oligomer as defined herein.

The invention also provides products (e.g. a pharmaceutical kit or acombined (“combination”) product) or compositions (e.g. a pharmaceuticalcomposition) wherein the product or composition comprises an alginateoligomer as herein defined and an antibiotic, e.g. selected from thegroup azithromycin, clarithromycin, dirithromycin, erythromycin,troleandomycin, aztreonam, imipenem, meropenem, ertapenem, doripenem,panipenem/betamipron, biapenem, PZ-601, cefixime, cefdinir, cefditoren,cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten,ceftizoxime, ceftriaxone, cefepime, demeclocycline, doxycycline,minocycline, oxytetracycline, tetracycline, bacitracin, colistin,polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin,lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, and trovafloxacin.Preferably the antibiotic is selected from the group ceftazidime,imipenem/cilastatin, meropenem, aztreonam, oxytetracycline, colistin,azithromycin and ciprofloxacin, preferably it is azithromycin. Forexample, the antibiotic may be selected from amikacin, gentamicin,kanamycin, neomycin, netilmicin, streptomycin, tobramycin, azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin,telithromycin, CarbomycinA, josamycin, kitasamycin, midecamicine,oleandomycin, spiramycin, tylosin, troleandomycin, aztreonam, imipenem,meropenem, ertapenem, doripenem, panipenem/betamipron, biapenem, PZ-601,cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime,ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime,demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline,bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,ofloxacin, and trovafloxacin. In particular, antibiotic may selectedfrom ceftazidime, imipenem/cilastatin, meropenem, aztreonam,oxytetracycline, azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, spiramycin and ciprofloxacin, and it isparticularly preferred that the antibiotic is selected from ceftazidime,imipenem/cilastatin, meropenem, aztreonam, azithromycin, clarithromycin,dirithromycin, erythromycin, roxithromycin, spiramycin andciprofloxacin. More preferably the antibiotic is selected fromaztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, spiramycin and ciprofloxacin. In other embodiments theantibiotic used is not tobramycin, amikacin and/or colistin. In otherembodiments the antibiotic used is not an aminoglycoside or apolypeptide antibiotic. In other embodiments the antibiotic used is notan antibiotic that has a positive charge under the conditions in whichit will be used with the alginate oligomer, e.g. antibiotics with atleast 3, e.g. at least 4, 5, 6 or 7 amino (—NH₂) groups. These productsand compositions are specifically contemplated as for use in the methodsof the invention. The products and compositions can be pharmaceutical ornon-pharmaceutical. Therefore the products and compositions of thisaspect of the invention can be used in any of the methods of theinvention.

As discussed above, the alginate oligomers and the antibiotics proposedfor use according to the invention may be used in combination with eachother, for example to be administered together, in a singlepharmaceutical formulation or composition, or separately (i.e. forseparate, sequential or simultaneous administration). Thus, the alginateoligomers and the antibiotics of the invention may be combined, e.g. ina pharmaceutical kit or as a combined (“combination”) product.

Thus as noted above, further aspects of the present invention provideproducts containing an alginate oligomer and an antibiotic as a combinedpreparation for the uses defined herein. Such products may optionallyfurther contain a further active agent.

The use of alginate oligomers as herein defined to manufacture suchpharmaceutical products and pharmaceutical compositions for use in themedical methods of the invention is also contemplated.

Further active agents may also be incorporated. The above and followingdiscussion of additional active agents and excipients and the like isdirectly applicable in its entirety to this aspect of the invention.

The active ingredient may be incorporated, optionally together withother active agents, with one or more conventional carriers, diluentsand/or excipients, to produce conventional galenic preparations such astablets, pills, powders (e.g. inhalable powders), lozenges, sachets,cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols(as a solid or in a liquid medium), sprays (e.g. nasal sprays),compositions for use in nebulisers ointments, soft and hard gelatinecapsules, suppositories, sterile injectable solutions, sterile packagedpowders, and the like. Sterile inhalable compositions are of particularnote for use in the treatment of respiratory diseases associated withMDR bacteria (which may include COPD, COAD, COAP, pneumonia, cysticfibrosis, emphysema and asthma).

Examples of suitable carriers, excipients, and diluents are lactose,dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calciumphosphate, inert alginates, tragacanth, gelatine, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, watersyrup, water, water/ethanol, water/glycol, water/polyethylene,hypertonic salt water, glycol, propylene glycol, methyl cellulose,methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesiumstearate, mineral oil or fatty substances such as hard fat or suitablemixtures thereof. Excipients and diluents of note are mannitol andhypertonic salt water (saline).

The compositions may additionally include lubricating agents, wettingagents, emulsifying agents, suspending agents, preserving agents,sweetening agents, flavouring agents, and the like. Additionaltherapeutically active agents may be included in the pharmaceuticalcompositions, as discussed above in relation to combination therapiesabove.

In some instances it may be beneficial to administer the alginateoligomers and/or the antibiotics as defined herein to animals, e.g. topromote weight gain/growth. Administration can be achieved in the formof the pharmaceutical compositions described above, but conveniently thealginate oligomers and/or the antibiotics as defined herein may be usedas a conventional feed additive, i.e. a compound that is added to animalfeed in small, nutritionally inconsequential amounts. The use of feedadditives in animal feeds is well established and it would be entirelyroutine for a skilled man to determine and use appropriate amounts ofthe alginates of the invention to achieve the desired effects, e.g.weight gain/growth.

The relative content of the alginate oligomer and the antibiotic canvary depending on the dosage required and the dosage regime beingfollowed and this will depend on the subject to be treated and thelocation and identity of the MDR bacterium, and/or the constituents ofthe contamination or population comprising the MDR bacterium.Preferably, the composition will comprise an amount of alginate oligomerand an amount of antibiotic that will provide a measurable reduction inthe resistance (or measurable increase in susceptibility or measurabledecrease in tolerance) to the antibiotic displayed by the bacterium e.g.an amount of alginate oligomer that will at least double, at leastquadruple, at least octuple, at least sexdecuple or at leastduotrigecuple the susceptibility of the MDR bacterium, to theantibiotic. Put in a different way, the composition will comprise anamount of alginate oligomer and an amount of antibiotic that willprovide a measurable treatment of the infection being targeted.Preferably the composition or product will comprise sufficient alginateoligomer that upon administration to a subject or application to alocation, the local concentration of the oligomer will be at least 2%,preferably at least 4%, 6% or 8% and most preferably at least 10%(weight by volume). The antibiotic preferably will be present in anamount that is sufficient to provide a local concentration of at least0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 64, 128, 256, 512,1024, 2048 or 4096 μg/ml. The skilled man would know that the amounts ofalginate oligomer and/or antibiotic can be reduced if a multiple dosingregime is followed or increased to minimise the number ofadministrations or applications.

The compositions and products of this aspect will typically comprisebetween 1% and 99%, 5% and 95%, 10% and 90% or 25% and 75% alginateoligomer and 1% and 99%, 5% and 95%, 10% and 90% or 25% and 75%antibiotic, allowance being made for other ingredients.

Parenterally administrable forms, e.g., intravenous solutions, should besterile and free from physiologically unacceptable agents, and shouldhave low osmolarity to minimize irritation or other adverse effects uponadministration and thus solutions should preferably be isotonic orslightly hypertonic, e.g. hypertonic salt water (saline). Suitablevehicles include aqueous vehicles customarily used for administeringparenteral solutions such as Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection,Lactated Ringer's Injection and other solutions such as are described inRemington's Pharmaceutical Sciences, 15th ed., Easton: Mack PublishingCo., pp. 1405-1412 and 1461-1487 (1975) and The National Formulary XIV,14th ed. Washington: American Pharmaceutical Association (1975). Thesolutions can contain preservatives, antimicrobial agents, buffers andantioxidants conventionally used for parenteral solutions, excipientsand other additives which are compatible with the biopolymers and whichwill not interfere with the manufacture, storage or use of products.

For topical administration the alginate oligomer and/or the antibioticcan be incorporated into creams, ointments, gels, transdermal patchesand the like. The alginate oligomers and/or the antibiotic can also beincorporated into medical dressings, for example wound dressings e.g.woven (e.g. fabric) dressings or non-woven dressings (e.g. gels ordressings with a gel component). The use of alginate polymers indressings is known, and such dressings, or indeed any dressings, mayfurther incorporate the alginate oligomers of the invention.

Accordingly, in a further specific embodiment, the invention furtherprovides a wound dressing comprising an alginate oligomer (which may beany alginate oligomer as herein defined) and/or an antibiotic (which maybe any antibiotic as herein defined) for use, where appropriate, in thetreatments and methods of the invention.

Further topical systems that are envisaged to be suitable are in situdrug delivery systems, for example gels where solid, semi-solid,amorphous or liquid crystalline gel matrices are formed in situ andwhich may comprise the alginate oligomer and/or the antibiotic. Suchmatrices can conveniently be designed to control the release of thealginate oligomer and/or the antibiotic from the matrix, e.g. releasecan be delayed and/or sustained over a chosen period of time. Suchsystems may form gels only upon contact with biological tissues orfluids. Typically the gels are bioadhesive. Delivery to any body sitethat can retain or be adapted to retain the pre-gel composition can betargeted by such a delivery technique. Such systems are described in WO2005/023176.

For application to oral, buccal and dental surfaces, toothpastes, dentalgels, dental foams and mouthwashes are mentioned specifically. Thus, inone particular aspect is included an oral health care, or oral hygiene,composition, comprising an alginate oligomer and an antibiotic (whichmay be any alginate oligomer or antibiotic as defined herein),particularly a mouthwash, toothpaste, dental gel or dental foam for use,where appropriate, in the treatments and methods of the invention.

Inhalable compositions are also of note. The formulation of compositionssuitable for inhalation is routine for the skilled man and has long beenstandard practice in the treatment of respiratory diseases. Inhalablecompositions may, for instance, take the form of inhalable powders,solutions or suspensions. The skilled man would be able to select themost appropriate type of delivery system for his needs and be able toprepare a suitable formulation of the alginates and/or antibiotics ofthe invention for use in that system. Propellant-free nebulisablesolutions and inhalable powder formulations are particularly preferred.

As noted above, a preferred composition of the invention is adebridement composition that is used in a debridement process to removea colony or population comprising an MDR bacterium, for example from atissue. Typically such a composition will be liquid, but gels, gel-sols,or semi-solid compositions might be used. The composition might be usedto debride the colony/population (e.g. by application to the tissueunder pressure) and/or may be used to bathe the tissue before, duringand/or after debridement by other means such as by surgical, mechanicalor chemical processes. The skilled person is readily able to formulatedebridement compositions in accordance with the invention.

In the case of an MDR bacterium on an inanimate surface on in aninanimate material, the alginate oligomer and/or antibiotic may beapplied to the surface or material to be treated in any convenientcomposition or formulation, or by any convenient means. Thus thealginate oligomer and/or antibiotic may be in liquid, gel, gel-sol,semi-solid or solid form (e.g. solutions, suspensions, homogenates,emulsions, pastes, powders, aerosols, vapours). Typically thecompositions for treating such inanimate surfaces or materials will be anon-pharmaceutically acceptable composition. The choice of compositionform will be dictated by the identity of the MDR bacterium on thesurface or in the material and location of the surface or material. Forinstance, if the location is a fluid line it might be convenient toapply a fluid composition. It might also be preferred to use acomposition that persists on the surface or in the part of the fluidline to be treated but that will not leach into the fluid of normal use,e.g. an adhesive gel. The skilled person is readily able to preparesuitable compositions from his common general knowledge. For instance,the alginate oligomer and/or antibiotic may be added to a paintformulation and applied to the surface to be treated, e.g. a boat hullor other part of a boat's structure that is exposed to water, or to abuilding or any part thereof, a tank (e.g. a storage or processing tank)or indeed to any part of any industrial machinery. Such compositions mayconveniently also comprise a further anti-microbial agent, as describedabove, e.g. an antibiotic, chlorine bleach, TCP, ethanol, Virkon™,povidone-iodine, silver compounds, antimicrobial surfactants, etc. Asthe compositions need not be pharmaceutically acceptable, harsherantimicrobials can be used subject to considerations of surface damage,environmental contamination, user safety and contamination of thetreated surface and interaction with the other components of thecomposition.

The compositions of the invention may be formulated so as to providequick, sustained or delayed release of the active ingredient afteradministration to the subject/surface by employing procedures well knownin the art. Adhesive compositions are also preferred. Adhesive,sustained and/or delayed release formulations may be particularlyconvenient.

In a further aspect the invention provides products susceptible tocontamination/colonisation by MDR bacteria whose susceptible surfaceshave been pretreated with an alginate oligomer and an antibiotic asdefined herein.

By “pretreated” it is meant that the susceptible surface is exposed toan alginate oligomer and/or an antibiotic prior to an exposure to an MDRbacterium and that the alginate oligomer and/or antibiotic persists onthe surface for a duration sufficient to preventcontamination/colonisation by an MDR bacterium for an appreciableduration of time. Preferably the alginate oligomer and/or the antibioticwill persist for substantially the useful life of the surface, e.g. thepretreatment results in a substantially permanent coating of an alginateoligomer and/or an antibiotic. Thus a pre-treated surface/product is oneto which the alginate olgimer and/or antibiotic is applied and on whichit remains. Such a product/surface may be a coated product/surface.

Non-limiting examples of products and surfaces susceptible tocontamination/colonisation by MDR bacteria are described above.Particular mention may be made of medical devices (e.g. endotracheal ortracheostomy tubes) and food or drink processing, storage or dispensingequipment. Pretreatment can be achieved by any convenient means, forexample any form of applying the alginate oligomer and/or antibiotic tothe surface, notably coating the surface, e.g. spray drying, polymercoating with a polymer incorporating the alginate oligomer and/orantibiotic, and painting, varnishing or lacquering with paint, varnishor lacquer formulations containing the alginate oligomer and/orantibiotic. Such a “coating” composition (e.g. a paint, varnish orlacquer) containing an alginate oligomer and/or antibiotic represents afurther aspect of the present invention. Alternatively, the alginateoligomer and/or antibiotic can be incorporated into the material fromwhich the object or its susceptible parts are manufactured. Thisapproach is suited to objects, or constituent parts thereof,manufactured from polymers such as plastics and silicones, e.g. themedical and surgical devices described above. Products comprising aninanimate surface comprising an alginate oligomer and/or antibioticcoating or coating composition, or incorporating an alginate oligomerand/or antibiotic are therefore contemplated. Non-limiting examples ofsuch products and surfaces are described above. Of particular note aremedical and surgical devices. This may include any kind of line,including catheters (e.g. central venous and urinary catheters),prosthetic devices e.g., heart valves, artificial joints, false teeth,dental crowns, dental caps and soft tissue implants (e.g. breast,buttock and lip implants). Any kind of implantable (or “in-dwelling”)medical device is included (e.g. stents, intrauterine devices,pacemakers, intubation tubes (e.g. endotracheal or tracheostomy tubes),prostheses or prosthetic devices, lines or catheters). Further productsinclude food processing, storage, dispensing or preparation equipment orsurfaces, tanks, conveyors, floors, drains, coolers, freezers, equipmentsurfaces, walls, valves, belts, pipes, air conditioning conduits,cooling apparatus, food or drink dispensing lines, heat exchangers, boathulls or any part of a boat's structure that is exposed to water, dentalwaterlines, oil drilling conduits, contact lenses and storage cases.

The invention will be further described with reference to the followingnon-limiting Examples.

EXAMPLES Example 1 Effect of G-Block Alginate Oligomers on the MinimumInhibitory Concentrations of Various Antibiotics for Various BacterialStrains

Materials and Methods

Bacterial Strains Used:

-   -   PA01 Pseudomonas aeruginosa ATCC 15692    -   Pseudomonas aeruginosa ATCC 39324, mucoid type strain (R79)*    -   Pseudomonas aeruginosa CFA 24-1, clinical mucoid strain (R80)*    -   Pseudomonas aeruginosa MDR R22 from China (V1)*    -   Pseudomonas aeruginosa MDR 301 from Poland (V2)*    -   Klebsiella pneumoniae KP05 506 from India (V3)*    -   Acinetobacter baumannii MDR ACB from Libya (V4)*

*Non-official labels assigned for internal identification purposes only.

Abbreviations used: Pseudomonas aeruginosa, (PA); Klebsiella pneumoniae(KP); Acinetobacter baumannii (ACB)

Media and Bacterial Strains Used:

Following retrieval from −80° C. storage, bacterial colonies were grownon blood agar with 5% sheep blood and were used to inoculate tryptonesoya broth (TSB) for overnight growth. Antibiotics were diluted incation-adjusted Mueller-Hinton broth (CAMHB) or CAMHB with G-fragments(Oligo CF-5/20 90-95% G residues) at 2%, 6% or 10%. Antibiotics werepharmaceutical grade purchased from Sigma-Aldrich. OligoG CF-5/20G-fragments were provided by Algipharma AS, Norway.

Minimum Inhibitory Concentration assay (Jorgensen et al., Manual ofClinical Microbiology, 7th ed. Washington, D.C.: American Society forMicrobiology, 1999; 1526-43):

Overnight bacterial cultures as described above were diluted in sterilewater until the OD625 was between 0.08 and 0.10 to confirm that the celldensity was equivalent to 0.5 McFarland standard.

In experiments with single antibiotics, two-fold antibiotic serialdilutions were prepared in CAMHB or CAMHB supplemented G-fragments(Oligo CF-5/20 90-95% G residues) at 0%, 2%, 6% or 10% and were placedin duplicate wells of flat-bottom 96-well microtiter plates (100 μl ineach well).

In experiments with two antibiotics (ceftazidime and azithromycin orciprofloxacin and azithromycin), two-fold antibiotic serial dilutionswere prepared in CAMHB or CAMHB supplemented with azithromycin at either1, 2, 4, or 8 μg/ml and G-fragments at either 0%, 2%, 6% or 10% and wereplaced in duplicate wells of flat-bottom 96-well microtiter plates (100μl in each well).

Bacterial cultures at 0.5 McFarland standard were diluted ten-fold inCAMHB and 5 μl added to the microtiter plates containing the antibioticserial dilutions. Plates were wrapped in parafilm and incubated at 37°C. for 16-20 hours. MIC values for each antibiotic/antibioticcombination were determined as the lowest concentration at which therewas no visible growth. Results are shown in Tables 1, 2 and 3.

TABLE 1 Minimum inhibitory concentration (MICs) of different antibioticsfor different Pseudomonas aeruginosa, Klebsiella pneumoniae andAcinetobacter baumannii strains in the presence of varyingconcentrations of OligoCF-5/20 (0-10%). (MIC values are expressed in μgml⁻¹).

TABLE 2 Minimum inhibitory concentration (MICs) of azithromycin for anMDR Acinetobacter baumannii and various strains of Pseudomonasaeruginosa and Klebsiella pneumoniae in the presence of varyingconcentrations of OligoCF-5/20 (0-10%).

TABLE 3 Minimum inhibitory concentrations (MICs) of two antibiotics incombinations with each other (azithromycin with either ceftazidime orciprofloxacin) for multi drug resistant (MDR) strains of Pseudomonasaeruginosa and Acinetobacter baumannii in the presence of varyingconcentrations of OligoCF-5/20 (0-10%) (MIC values are expressed in μgml⁻¹).

Results and Discussion

In general, treatment of planktonically growing MDR strains ofPseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacterbaumannii strains with increasing concentrations of OligoG CF-5/20lowered the MIC values of the antibiotics used (Tables 1, and 2).Oxytetracycline, azithromycin and ciprofloxacin were all shown to havedecreasing MICs with increasing amounts of OligoG CF-5/20 used. Thus, inthe case of these antibiotics, the data appear to show that alginateoligomers may potentiate their effects. The antibiotics tested includeantibiotics common in the treatment of cystic fibrosis.

The magnitude of the effect was most pronounced for the MDR strainPseudomonas aeruginosa strain R22, although all strains studiedresponded to treatment with the alginate oligomers and azithromycin. Theresults also show alginate oligomers potentiate the antibioticazithromycin with all strains tested. Such an effect may be seen withazithromycin alone or in combination with other antibiotics.

More specifically, for the MDR Pseudomonas strains, primaxin (acombination of imipenem and cilastatin), azithromycin, ceftazidime,ciprofloxacin and aztreonam were all more effective when used incombination with the alginate oligomers. Two antibiotics in conjunctionwith alginate oligomers were more effective against KP05 506, namely,azithromycin and aztreonam, but the data from experiments using primaxinand meropenem is inconclusive. In combination with alginate oligomers,azithromycin, ceftazidime, ciprofloxacin and aztreonam showed a morepositive effect on the Acinetobacter baumannii isolate.

The effects of azithromycin in conjunction with either ceftazidime orciprofloxacin in the presence of alginate oligomers on the MDR R22 PAstrain and the MDR Acinetobacter baumannii isolate were tested and theresults can be seen in Table 3. In all cases MIC values of theceftazidime or ciprofloxacin in the antibiotic combinations were reducedby various concentrations of alginate oligomer.

Example 2

The study described in Example 1 was repeated with the following strainsof bacteria and antibiotics as detailed in Tables 4, 5 and 6.

Bacterial Strains

-   -   PA01 Pseudomonas aeruginosa ATCC 15692 (E77)    -   R79* Mucoid Pseudomonas aeruginosa ATCC 39324 ISOLATION: sputum        from a cystic fibrosis patient, Boston, Mass.    -   R80* Mucoid Pseudomonas aeruginosa CFA 24-1 (CLINICAL ISOLATE        from a CF patient.)    -   V1* R22 PSA (China) Pseudomonas aeruginosa    -   V2* MDR 301 PSA (Poland) Pseudomonas aeruginosa    -   V3* KP05 506 (India) Klebsiella pneumoniae.    -   V4* MDR ACB (Libya) Acinetobacter baumannii    -   V5* AIM-1 E. coli    -   V9* (Egypt) Acinetobacter baumannii    -   V10* (Egypt) Acinetobacter lwoffii    -   V11* 5702 (Wales) E. coli    -   V12* 5725 (Wales) Klebsiella pneumoniae    -   V22* 6056 Acinetobacter    -   V23* 1322 Burkholderia cepacia

*Non-official labels assigned for internal identification purposes only.

TABLE 4 Minimum inhibitory concentration (MICs) of different macrolideantibiotics for various strains of Pseudomonas aeruginosa, Klebsiellapneumoniae, Acinetobacter baumannii and E. coli displaying MDRphenotypes in the presence of varying concentrations of OligoCF-5/20(0-10%). (MIC values are expressed in μg ml⁻¹).

TABLE 5 Minimum inhibitory concentration (MICs) of different antibioticsfor strains of Burkholderia cepacia, Klebsiella pneumoniae,Acinetobacter baumannii, Acinetobacter lwoffii and E. coli displayingMDR phenotypes in the presence of varying concentrations of OligoCF-5/20(0-10%). (MIC values are expressed in μg ml⁻¹).

TABLE 6 Minimum inhibitory concentration (MICs) of different antibioticsfor a strain (V23) of Burkholderia cepacia in the presence of varyingconcentrations of OligoCF-5/20 (0-10%). (MIC values are expressed in μgml⁻¹). Results from three separate experiments.

In general, Table 4 validates the results disclosed in Tables 1 and 2 inrelation to the effects of OligoG CF-5/20 on the MIC's of macrolideantibiotics in a variety of planktonically growing bacteria. Invirtually every combination of bacteria and macrolide, MIC values arereduced by increasing concentrations of OligoG CF-5/20. The results alsoshow alginate oligomers potentiate the effects of the macrolideantibiotics with all bacteria tested. Such an effect may be seen withazithromycin alone or in combination with other antibiotics.

From data presented in Tables 4, 5 and 6 it can been seen that ingeneral increasing concentrations of OligoG CF-5/20 lowered the MICvalues of the antibiotics used against MDR strains of Pseudomonasaeruginosa, Klebsiella pneumoniae, Burkholderia cepacia, Acinetobacterlwoffii, Acinetobacter baumannii and E. coli. The antibiotics testedinclude antibiotics common in the treatment of cystic fibrosis.Aztreonam, primaxin (a combination of imipenem and cilastatin),ciprofloxacin, meropenem, ceftazidime, azithromycin, erythromycin,clarithromycin, and spiramycin were all shown to have decreasing MICswith increasing amounts of OligoG CF-5/20 used. Thus, in the case ofthese antibiotics, the data appear to show that alginate oligomers maypotentiate their effects. The macrolides display the greatest reductionin MICs with increasing amounts of OligoG CF-5/20 used. The magnitude ofthe effect was most pronounced for the Burkholderia tested andAcinetobacter baumannii strain V9 and in these strains every antibiotictested showed a reduction in MIC values with increasing concentrationsof alginate oligomer.

Table 6 further validates the results with Burkholderia presented inTable 5. This antibiotic potentiating effect seen with alginateoligomers and Burkholderia is of clinical significance as theseorganisms are associated with human and animal disease and are difficultto treat on account of their tendency to display antibiotic resistance.

Example 3

The study described in Example 1 was repeated with the following strainsof Acinetobacter baumannii, antibiotics and M-block alginate oligomer inplace of OligoG CF-5/20 as detailed in Table 7. The M-block oligomer is100% M with a DPn of 15 to 18.

TABLE 7 Minimum inhibitory concentration (MICs) of different antibioticsfor a strain of Acinetobacter baumannii displaying an MDR phenotype anda strain of Acinetobacter baumannii displaying an non-MDR phenotype inthe presence of varying concentrations of M-block oligomer (0-10%). (MICvalues are expressed in μg ml⁻¹).

The results displayed in Table 7 show that M-block oligomers are, likeOligoG CF-5/20, also effective in lowering MIC values for a number ofdifferent antibiotics (including a macrolide) in MDR and non-MDR strainsof Acinetobacter baumannii.

Example 4

Further MIC assays were conducted with the various strains andantibiotics recited in Tables 8 to 11 using the following protocol.

MIC-Assay

G-block alginates (OligoG CF-5/20) were dissolved in Mueller-Hintonbroth (Lab M limited, LAB114 Mueller-Hinton broth) to 1.25 times of thedesired assay concentrations (2, 6 and 10%). Antibiotics were dissolvedin Mueller-Hinton broth and Mueller-Hinton broth with G-block alginateat a concentration of 1.25 times the highest desired assayconcentrations. Antibiotics were pharmaceutical grade purchased fromSigma-Aldrich. OligoG CF-5/20 G-fragments were provided by AlgipharmaAS, Norway.

Two-fold serial dilutions of antibiotics were made in Mueller-Hintonwith different concentrations of G-block alginate, and the solutionswere placed in four parallel wells in Nunc 384-well micro plates (30 μlper well in Nunc 242757 microplates). A group of 8 wells with noaddition of antibiotics for each G-block concentration was included oneach micro plate as growth reference.

Frozen stock cultures were made from over night cultures in TSB-brothfor all strains by addition of glycerol to 15% concentration prior tofreezing at −80° C. At the day of analysis, overnight TSB cultures (6 mlin 50 ml tube tilted to 45-degrees angle, 200 rpm, 2.5 cm amplitude, 37°C.) were diluted in TSB until the OD600 was 0.10, and further diluted1:40 in Mueller-Hinton broth. Each well in the 384-well assay plates wasinoculated with 7.5 μl of the diluted culture. The microplates wereplaced in plastic bags and incubated at 37° C. The optical density at600 nm in the microwells was measured after approximately 18 hours ofincubation, and the relative growth yield in each well was calculatedbased on the growth in the reference groups. The MIC value was set tothe highest concentration giving less than 30% growth in all 4 parallelwells within the sample groups. The microplates were further incubatedfor 8 hours, and optical density in the cultures was measured once morefor confirmation of the estimated MIC-values.

Results

In each of Tables 8 to 11 there is a main table of basic data, and asecondary table which is a representation of the overall effect of theOligoCF-5/20 on the MIC value for each particular bacteria andantibiotic combination. In the secondary table a dark shaded boxrepresents an overall reduction in the MIC value; a hatched boxedrepresents an overall increase in the MIC value; M indicates that all ofthe MIC values were greater than the maximum concentration of antibioticused; L indicates that all of the MIC values were less than the minimumconcentration of antibiotic used; NE indicates no effect on the MICvalues was observed; ND indicates that the particular combination ofantibiotic and bacteria was not tested.

Table 8. Minimum inhibitory concentration (MICs) of differentantibiotics for strains of Burkholderia cepacia and Pseudomonasaeruginosa displaying MDR phenotypes in the presence of varyingconcentrations of OligoCF-5/20 (0-10%). (MIC values are expressed in μgml⁻¹).

Table 9. Minimum inhibitory concentration (MICs) of differentantibiotics for strains of Acinetobacter baumannii and Acinetobacterlwoffii displaying MDR phenotypes in the presence of varyingconcentrations of OligoCF-5/20 (0-10%). (MIC values are expressed in μgml⁻¹).

Table 10. Minimum inhibitory concentration (MICs) of differentantibiotics for strains of Klebsiella pneumoniae displaying MDRphenotypes in the presence of varying concentrations of OligoCF-5/20(0-10%). (MIC values are expressed in μg ml⁻¹).

Table 11. Minimum inhibitory concentration (MICs) of differentantibiotics for strains of E. coli and Providencia stuartii displayingMDR phenotypes in the presence of varying concentrations of OligoCF-5/20(0-10%). (MIC values are expressed in μg ml⁻¹).

Table 12. Minimum inhibitory concentration (MICs) of differentantibiotics for strains of Streptococcus oralis and Staphylococcusaureus (MRSA) displaying MDR phenotypes in the presence of varyingconcentrations of OligoCF-5/20 (0-10%). (MIC values are expressed in μgml⁻¹).

TABLE 8

TABLE 9

TABLE 10

TABLE 11

TABLE 12

The data presented in Tables 8 to 12 generally show that increasingconcentrations of OligoCF-5/20 (0-10%) decreases MIC values for allantibiotics tested (azithromycin, erythromycin, roxithromycin,dirithromycin (macrolides) aztreonam (monobactam) ceftazidime(cephalosporin) imipenem (carbapenem), ciprofloxacin (quinolone) andoxytetracycline (tetracycline)) in one bacterial strain or another).Notably, Table 12 shows that OligoCF-5/20 reduces MIC values in Grampositive organisms (MRSA U50 and Streptococcus oralis). The effect isparticularly pronounced with the MRSA strain tested. This highlights thegeneral applicability of the use of alginate oligomers alongsideantibiotics in the treatment of all MDR bacteria (whether Gram negative,Gram positive, or Gram test non-responsive) e.g. by overcoming theresistance of MDR bacteria to antibiotic treatments or enhancing theefficacy of those antibiotics.

The effect was most consistently observed across the antibiotics testedin the Pseudomonas, Acinetobacter, Burkholderia and MRSA species tested,and strains V1, V2, V23, V4 and V9 in particular. Interestingly, in thisExample strains V23 and V9 showed five instances of NE (no effect) or M(MICs were above the maximum concentration of antibiotic used), howeverin Example 2, data shows that these five combinations of bacteria andantibiotic do in fact display reductions in MIC with increasingconcentrations of OligoCF-5/20. This highlights the more specificapplicability of the use of alginate oligomers alongside antibiotics inthe treatment of MDR Pseudomonas, Acinetobacter, Burkholderia and MRSA,e.g. by overcoming the resistance these bacteria have to antibiotictreatments or enhancing the efficacy of those antibiotics against thesebacteria in particular.

The effect is most consistently observed across the strains tested withthe macrolides (azithromycin, erythromycin, roxithromycin,dirithromycin) and to a slightly lesser extent aztreonam, ceftazidimeand ciprofloxacin. This highlights the more specific applicability ofalginate oligomers to the treatment of bacteria in general, includingMDR bacteria with macrolides (e.g. azithromycin, erythromycin,roxithromycin, dirithromycin) in particular, but also quinolones (e.g.ciprofloxacin), monobactams (e.g. aztreonam) and cephalosporins (e.g.ceftazidime), e.g. by overcoming the resistance in bacteria to theseantibiotic treatments or by enhancing the efficacy of these antibioticsagainst bacteria.

Also of significant note is the evidence provided in Table 11 that showsthat OligoCF-5/20 can lower MIC values for a β-lactam (imipenem, acarbapenem) in MDR Providencia stuartii. β-lactam resistance inProvidencia populations is rising and so alginate oligomers mayrepresent a new approach to the treatment of Providencia infections

The invention claimed is:
 1. A method of overcoming resistance to atleast one antibiotic in a Gram negative, multidrug resistance (MDR)bacterium, said method comprising contacting said bacterium with analginate oligomer having an average molecular weight of less than 30,000Daltons together with the at least one antibiotic, wherein theresistance to at least one antibiotic is overcome or reduced, whereinthe Gram negative MDR bacterium is initially resistant to at least threeclasses of antibiotics and wherein said initial resistance to at leastthree classes of antibiotics is seen in a bacterium that is not in abiofilm mode of growth.
 2. The method of claim 1, said method comprisingadministering to a subject infected, suspected to be infected, or atrisk of infection with a Gram negative MDR bacterium said alginateoligomer together with the at least one antibiotic to overcomeresistance to the at least one antibiotic in said bacterium.
 3. Themethod of claim 2 comprising separate, simultaneous or sequentialadministration of said alginate oligomer together with the at least oneantibiotic to a subject infected, suspected to be infected, or at riskof infection, with a Gram negative MDR bacterium.
 4. The method of claim1, said method comprising contacting a site and/or said bacteria withsaid alginate oligomer together with the at least one antibiotic towhich said bacteria are resistant.
 5. The method of claim 1, wherein thebacterium is resistant to three or more classes of antibiotics selectedfrom the group consisting of macrolides, β-lactams, tetracyclines,polypeptide antibiotics and quinolones.
 6. The method of claim 5 whereinthe bacterium is resistant to three or more antibiotics selected fromthe group consisting of azithromycin, clarithromycin, dirithromycin,erythromycin, troleandomycin, roxithromycin, spiramycin, aztreonam,imipenem, meropenem, ertapenem, doripenem, panipenem/betamipron,biapenem, PZ-601, cefixime, cefdinir, cefditoren, cefoperazone,cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime,ceftriaxone, cefepime, demeclocycline, doxycycline, minocycline,oxytetracycline, tetracycline, bacitracin, colistin, polymyxin B,ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin,moxifloxacin, norfloxacin, ofloxacin, and trovafloxacin.
 7. The methodof claim 1, wherein the bacterium is resistant to one or moreantibiotics selected from the group consisting of ceftazidime,imipenem/cilastatin, meropenem, aztreonam, azithromycin, clarithromycin,dirithromycin, erythromycin, roxithromycin, spiramycin, oxytetracyclineand ciprofloxacin.
 8. The method of claim 1, wherein the bacterium isresistant to one or more antibiotics that is a conventional treatmentfor that bacterium.
 9. The method of claim 1, wherein the bacterium isfrom the family Enterobacteriacee or is a non-fermenting Gram negativebacterium.
 10. The method of claim 9, wherein the bacterium is selectedfrom the genera consisting of Pseudomonas, Acinetobacter,Stenotrophomonas, Burkholderia, Escherichia, Providencia and Klebsiella.11. The method of claim 1, wherein the bacterium is an MDR strainselected from the group consisting of Pseudomonas aeruginosa, Klebsiellapneumoniae, Burkholderia cepacia, Providencia stuartii or Acinetobacterbaumannii that is resistant to one or more antibiotics selected from thegroup consisting of ciprofloxacin, meropenem, ceftazidime, aztreonam,azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, spiramycin, oxytetracycline and imipenem/cilastatin. 12.The method of claim 1, wherein the at least one antibiotic is selectedfrom the group consisting of macrolides, β-lactams, tetracyclines,polypeptide antibiotics and quinolones.
 13. The method of claim 12,wherein the at least one antibiotic is selected from the groupconsisting of azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, telithromycin, CarbomycinA, josamycin, kitasamycin,midecamicine, oleandomycin, spiramycin, tylosin, troleandomycin,aztreonam, imipenem, meropenem, ertapenem, doripenem,panipenem/betamipron, biapenem, PZ-601, cefixime, cefdinir, cefditoren,cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten,ceftizoxime, ceftriaxone, cefepime, demeclocycline, doxycycline,minocycline, oxytetracycline, tetracycline, bacitracin, colistin,polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin,lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, and trovafloxacin.14. The method of claim 12, wherein the at least one antibiotic is amacrolide.
 15. The method of claim 1, wherein the bacterium isBurkholderia sp.
 16. The method of claim 15, wherein said Burkholderiasp. is selected from Burkholderia cepacia, Burkholderia pseudomallei andBurkholderia mallei.
 17. The method of claim 15, wherein said at leastone antibiotic is selected from the group consisting of azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin,telithromycin, CarbomycinA, josamycin, kitasamycin, midecamicine,oleandomycin, spiramycin, tylosin, troleandomycin, aztreonam, imipenem,meropenem, ertapenem, doripenem, panipenem/betamipron, biapenem, PZ-601,cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime,ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime,demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline,bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,ofloxacin, and trovafloxacin.
 18. The method of claim 1, wherein thebacterium is a clinical strain or a clinical isolate.
 19. The method ofclaim 1, wherein the alginate oligomer has an average molecular weightof less than 25,000 Daltons.
 20. The method of claim 1, wherein thealginate oligomer has a number average degree of polymerization of 2 to100.
 21. The method of claim 1, wherein the alginate oligomer has up to100 monomer residues.
 22. The method of claim 1, wherein the alginateoligomer has at least 70% G residues.
 23. The method of claim 22 whereinthe alginate oligomer has at least 80% G residues.
 24. The method ofclaim 22, wherein at least 80% of the G residues are arranged inG-blocks.
 25. The method of claim 1, wherein the alginate oligomer hasat least 70% M residues.
 26. The method of claim 25 wherein the alginateoligomer has at least 80% M residues.
 27. The method of claim 25,wherein at least 80% of the M residues are arranged in M blocks.
 28. Themethod of claim 2, wherein the infection is of an internal or externalbody surface selected from the group consisting of a surface in the oralcavity, the reproductive tract, the urinary tract, the respiratorytract, the gastrointestinal tract, the peritoneum, the middle ear, theprostate, vascular intima, the eye, including the conjunctiva or cornealtissue, lung tissue, heart valves, skin, scalp, nails, the interior ofwounds or the surface of adrenal, hepatic, renal, pancreatic, pituitary,thyroid, immune, ovarian, testicular, prostate, endometrial, ocular,mammary, adipose, epithelial, endothelial, neural, muscle, pulmonary,epidermis or osseous tissue; or in a body fluid selected from blood,plasma, serum, cerebrospinal fluid, GI tract contents, sputum, pulmonarysecretions and semen; or in or on body tissue selected from adrenal,hepatic, renal, pancreatic, pituitary, thyroid, immune, ovarian,testicular, prostate, endometrial, ocular, mammary, adipose, epithelial,endothelial, neural, muscle, pulmonary, epidermis and osseous tissue.29. The method of claim 2, wherein the subject is selected from thegroup consisting of a subject with a pre-established infection, animmunocompromised subject, a subject undergoing intensive or criticalcare, a subject suffering from trauma, a subject with a burn, a subjectwith an acute and/or chronic wound, a neonatal subject, an elderlysubject, a subject with cancer, a subject suffering from an auto-immunecondition, a subject with reduced or abrogated epithelial or endothelialsecretion and/or secretion clearance and a subject fitted with a medicaldevice.
 30. The method of claim 29, wherein the subject is selected fromthe group consisting of a subject with a condition selected from HIV,sepsis, septic shock, AIDS, a cancer of the immune system, rheumatoidarthritis, diabetes mellitus type I, Crohn's disease, COPD, COAD, COAP,bronchitis, cystic fibrosis, emphysema, lung cancer, asthma, pneumoniaand sinusitis, a subject preparing for, undergoing, or recovering fromchemotherapy and/or radiotherapy, an organ transplant subject and asubject resident in a healthcare institution or a smoker.
 31. The methodof claim 29, wherein the subject is selected from the group consistingof a subject with a respiratory condition or disease.
 32. The method ofclaim 1, wherein said bacterium is on an animate or inanimate surface orin an animate or inanimate material.
 33. The method of claim 1, whereinthe bacterium is on a surface selected from the group consisting ofsurfaces of food or drink processing, preparation, storage or dispensingmachinery or equipment, surfaces of air conditioning apparatus, surfacesof industrial machinery, surfaces of storage tanks, surfaces of medicalor surgical equipment, surfaces of aquatic/marine equipment or thesurfaces of buildings and other structures.
 34. The method of claim 33wherein the surface is selected from the group consisting of foodprocessing, storage, dispensing or preparation equipment or surfaces,tanks, conveyors, floors, drains, coolers, freezers, equipment surfaces,walls, valves, belts, pipes, air conditioning conduits, coolingapparatus, food or drink dispensing lines, heat exchangers, boat hulls,dental waterlines, oil drilling conduits, contact lenses, contact lensstorage cases, catheters, prosthetic devices and implantable medicaldevices.
 35. The method of claim 1, wherein the bacterium is in amaterial selected from the group consisting of clinical/scientificwaste, animal or human food stuffs, personal hygiene products,cosmetics, drinking water supplies, waste water supplies, agriculturalfeedstuffs and water supplies, insecticide formulations, pesticideformulations, herbicide formulations, industrial lubricants, cell andtissue culture media, and cell and tissue cultures.
 36. The method ofclaim 10, wherein the bacterium is one of Pseudomonas aeruginosa,Acinetobacter baumannii, Stenotrophomonas maltophilia, Burkholderia spp,E. coli, Providencia stuartii and Klebsiella pneumoniae.
 37. The methodof claim 20, wherein the alginate oligomer has a number average degreeof polymerization of 2 to
 35. 38. The method of claim 21, wherein thealginate oligomer is a 2- to 35-mer.
 39. The method of claim 31, whereinthe respiratory condition or disease is selected from the groupconsisting of COPD, COAD, COAP, bronchitis, cystic fibrosis, emphysema,lung cancer, asthma, and pneumonia.
 40. The method of claim 1, whereinthe alginate oligomer has at least 85% G residues.
 41. The method ofclaim 1, wherein the alginate oligomer has at least 90% G residues.